Development of an in vivo gene mutation assay using the endogenous Pig-A gene: II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing Pig-A cDNA from the mutants

We previously reported that rat spleen T-cells and peripheral red blood cells that are deficient in glycosylphosphatidylinositol (GPI) synthesis [presumed mutants for the phosphatidylinositol glycan complementation group A gene (Pig-A)] could be detected by flow cytometry (FCM) as cells negative for GPI-linked markers (CD48 and CD59, respectively). To establish this procedure as a rapid in vivo gene mutation assay, we have examined the Pig-A gene of GPI-deficient rat spleen T-cells for DNA sequence alterations. Splenocytes were isolated from male F344 rats, primed with ionomycin and phorbol-12-myristate-13-acetate, and seeded at limiting-dilution into 96-well plates. To select for GPI-deficient T-cells, the cells were cultured for 10 days in a medium containing rat T-STIM and 2 nM proaerolysin (ProAER). The frequency of ProAER-resistant (ProAER(r)) spleen T-cells from control rats ranged from 1.3 x 10(-6) to 4.8 x 10(-6), while administration of three doses of 40 mg/kg N-ethyl-N-nitrosourea increased the frequency of ProAER(r) T-cells 100-fold at 4 weeks after dosing. FCM analysis of the cells in ProAER(r) clones revealed that they were CD48-negative, and thus presumably GPI-deficient. Sequencing of Pig-A cDNA from six ProAER(r) clones indicated that they all contained alterations in the Pig-A protein coding sequence; five had base pair substitutions and one had multiple exons deleted. These results indicate that GPI-deficient spleen T-cells are Pig-A gene mutants and support the use of FCM analysis of GPI-deficient cells as a rapid assay for measuring in vivo gene mutation.     

In vivo mutation assay based on the endogenous Pig-a locus

The product of the X-chromosome's Pig-a gene acts in the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis, and is thereby essential for attaching certain proteins to the cell surface. The experiments described herein were designed to evaluate whether lack of GPI-anchored proteins could form the basis of an in vivo mutation assay. Specifically, we used a CD59-negative cell surface phenotype to denote Pig-a mutation. Besides anti-CD59-PE, two other fluorescent reagents were used: thiazole orange to differentiate mature erythrocytes, reticulocytes (RETs), and leukocytes; and anti-CD61 to resolve platelets. These experiments were performed with Sprague Dawley rats, and focused on two cell populations, total erythrocytes and RETs. The ability of the analytical method to enumerate CD59-negative erythrocytes was initially assessed with reconstruction experiments whereby mutant-mimicking cells were added to control bloods. Subsequently, female rats were treated on three occasions with the model mutagens ENU (100 mg/kg/day) or DMBA (40 mg/kg/day). Blood specimens were harvested at various intervals, as late as 6 weeks post-exposure. Considering all week 4-6 data, we found that CD59-negative cells ranged from 239 to 855 x 10(-6) and 82 to 405 x 10(-6) for ENU and DMBA, respectively. These values were consistently greater than those observed for negative control rats (18 +/- 19 x 10(-6)). The elevated frequencies observed for the genotoxicant-exposed animals were usually higher for RETs compared to total erythrocytes. These data support the hypothesis that an efficient in vivo mutation assay can be developed around flow cytometric enumeration of erythrocytes and/or RETs that exhibit aberrant GPI-anchored protein expression.     

Further development of the rat Pig-a mutation assay: measuring rat Pig-a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes

Recent studies indicate that the Pig-a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig-a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid-specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 x 10(6) CD71-positive RETs could be evaluated by flow cytometry for Pig-a mutant frequency within 5 to 8 min. CD59-deficient RET and BME frequencies of more than 100 x 10(-6) and 80 x 10(-6) were detected 1 week after treating rats with 40 mg/kg N-ethyl-N-nitrosourea; by comparison, the frequency of CD59-deficient total RBCs in these rats was 13.2 x 10(-6). The frequency of spontaneous Pig-a mutant RETs and BMEs was less than 5 x 10(-6) and 15 x 10(-6), respectively. Since approximately 98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59-deficient Pig-a mutations in cells of erythroid lineage. Conducting concurrent Pig-a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.     

Monitoring humans for somatic mutation in the endogenous PIG-a gene using red blood cells

The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10(-6) ; median of 3.8 × 10(-6) and mutant frequency less than 8 × 10(-6) for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ～300 × 10(-6) and ～100 × 10(-6) . We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ～6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine the assay's sensitivity in detecting mutations induced by genotoxic agents acting via different mechanisms.     

International Pig-a gene mutation assay trial (stage III): results with N-methyl-N-nitrosourea

N-methyl-N-nitrosourea (MNU) was evaluated in the in vivo Pig-a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28-day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low-dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig-a phenotypic mutation, CD59-negative reticulocytes/erythrocytes (RETs(CD592-)/ RBCs(CD592-)) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN-RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose-dependent increases in the frequency of RETs(CD592-) and RBCs(CD592-) were observed by Day 15 or 29, respectively. Dose-dependent increases were observed in %MN-RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig-a mutation assay results, consistent with previous observations with MNU and N-ethyl-N-nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.     

CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation.     

Analysis of mutations in the Pig-a gene of spleen T-cells from N-ethyl-N-nitrosourea-treated fisher 344 rats

A rapid in vivo somatic cell gene mutation assay is being developed that measures mutation in the endogenous X-linked phosphatidylinositol glycan, class A gene (Pig-a). The assay detects Pig-a mutants by flow cytometric identification of cells deficient in glycosylphosphatidyl inositol (GPI) anchor synthesis. GPI-deficient, presumed Pig-a mutant cells also can be detected in a cloning assay that uses proaerolysin (ProAER) selection. Previously, we demonstrated that ProAER-resistant (ProAER(r) ) rat spleen T-cells have mutations in the Pig-a gene. In the present study, we report on a more complete analysis of ProAER(r) rat spleen T-cell mutants and describe a mutation spectrum for mutants isolated from rats 4 weeks after treatment with three consecutive doses of 35.6 mg/kg N-ethyl-N-nitrosourea (ENU). We identified a total of 55 independent mutations, with the largest percentage (69%) involving basepair substitution at A:T. The overall spectrum of Pig-a gene mutations was consistent with the types of DNA adducts formed by ENU and was very similar to what has been described for in vivo ENU-induced mutation spectra in other rodent reporter genes (e.g., in the endogenous Hprt gene and transgenic shuttle vectors). These data are consistent with the rat Pig-a assay detecting test-agent-induced mutational responses.     

Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59

Exosomes are secreted nanometer-sized vesicles derived from antigen-presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell-free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen-presenting cell-derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)-anchored regulators CD55 and CD59, but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI-anchored complement regulators in order to permit their survival in the extracellular environment.     

International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.     

Analysis of immunoglobulin VH genes in CD10-positive diffuse large B-cell lymphoma

CD10, a proteolytic enzyme seen in germinal center cells and in the majority of follicular lymphomas, is occasionally expressed in diffuse large B-cell lymphomas (DLBCL). To clarify the origin and cellular characteristics of CD10-positive DLBCL, we analyzed 36 de novo cases of DLBCL for somatic mutations of the immunoglobulin heavy chain variable region (VH) genes and for their immunophenotypes. Expression greater than that of grade 2 Bcl-6 was observed in 11 of the 30 CD10-negative cases (37%) and in all six CD10-positive cases (100%; P < 0.05) without expression of CD5, CD23, cyclin D1, CD30 or CD138. The average mutation frequencies of the six CD10-positive and 30 CD10-negative DLBCL were 12.9 and 9.8%, respectively. The range of SM frequencies in CD10-positive DLBCL (9.52-18.06) was distinctly narrower than that observed for CD10-negative DLBCL (0.69-26.89). These findings seem to indicate that CD10-positive DLBCL, originating from germinal center B cells, is a genetically and immunophenotypically more homogeneous group than CD10-negative DLBCL. Furthermore, three extranodal lymphomas, in five of the six CD10-positive DLBCL, showed ongoing mutation, indicating that antigen-driven, high-affinity somatic mutation may play an important role in clonal expansion in CD10-positive DLBCL. All four extranodal cases of the six CD10-positive DLBCL showed ongoing mutation and/or bcl-2/JH rearrangement. This result suggests that the cell origin of extranodal CD10-positive DLBCL may be the same as that of follicular lymphomas.     

Report on stage III Pig-a mutation assays using benzo[a]pyrene

Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S. FDA-NCTR and Bayer-Germany. Starting on Day 1, groups of five 6- to 7-week-old male Fischer 344 (F344, used at FDA-NCTR) and Han Wistar rats (Bayer) were given 28 daily doses of 0, 37.5, 75, or 150 mg/kg BaP; blood was sampled on Days -1, 4, 15, 29, and 56. Pig-a mutant frequencies were determined on Days -1, 15, 29, and 56 in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies; percent micronucleated-RETs (%MN-RET) were measured on Days 4 and 29. RBC(CD59-) and RET(CD59-) frequencies increased in a dose- and time-dependent manner, producing significant increases by Day 29 in both rat models. The responses for RETs were stronger than those for RBCs, and the responses in F344 rats were stronger than in Han Wistar rats. BaP also produced significant increases in %MN-RET frequency at Days 4 and 29, with the responses being greater in F344 than Han Wistar rats. The overall findings were consistent with those of the reference laboratory using Han Wistar rats. Finally, mutation assays performed on splenocytes from Day 56 F344 rats indicated that BaP mutant frequencies were three to fivefold higher for the Hprt gene than the Pig-a gene. The results indicate that the Pig-a RET and RBC assays are reproducible, transferable, and show promise for integrating gene mutation into 28-day repeat-dose studies.     

Cell Activation Mediated by Glycosylphosphatidylinositol-Anchored or Transmembrane Forms of CD14

CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-κB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking.     

A regenerative erythropoietic response does not increase the frequency of Pig‐a mutant reticulocytes and erythrocytes in Sprague‐Dawley rats

The in vivo rodent Pig‐a mutation assay is a sensitive test to identify exposure to mutagenic substances, and has been proposed as an assay for the identification of impurities for pharmaceuticals. Red blood cells (RBCs) and reticulocytes (RETs) are analyzed by flow cytometry after exposure to potentially mutagenic chemicals for cells deficient in the cell surface anchored protein CD59, representing mutation in the X‐linked Pig‐a gene. The full potential of the assay as well as its limitations are currently being explored. The current study investigated the effects of regenerative erythropoietic bone marrow responses on the frequency of Pig‐a mutated reticulocytes (RET^CD59‐) and erythrocytes (RBC^CD59‐). We hypothesized that a robust regenerative erythropoietic response would not increase the basal frequency of RET^CD59‐ or RBC^CD59‐ cells. Two groups of six male Sprague‐Dawley rats either had 2 mL of blood sampled each day via an indwelling catheter over a period of 5 days or were minimally sampled for hematology and used as controls. Blood was also then collected and evaluated 5, 18, and 49 days after the initial bleed period for the number of Pig‐a mutant cells in either the RET or RBC population. Despite the expected decrease in hematocrit and the correlative increase in reticulocytes after bleeding, no increase in the number of Pig‐a mutant cells was observed in male Sprague‐Dawley rats that were bled for five consecutive days. These results indicate that changes in erythropoiesis and hematology parameters in rats appear to have no effect on the background levels of Pig‐a mutated RETs and RBCs. Environ. Mol. Mutagen. 59:91–95, 2018. © 2017 Wiley Periodicals, Inc.     

A gel microtyping system for diagnosis of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH), an acquired stem cell defect, is underdiagnosed because of its atypical symptoms in some patients and because available methods, which are time consuming and complicated, are not widely used. The hemolysis of PNH red blood cells (RBCs) is attributed to their enhanced susceptibility to complement lysis caused by a deficiency in glycosylsphosphatidylinositol (GPI)-anchored complement regulatory membrane proteins, especially membrane inhibitor of reactive lysis (MIRL [CD59]). We evaluated the diagnostic value of a simple hemagglutination test using the gel microtyping system by comparing it with lytic tests (the Ham test and the sucrose lysis test) and with flow cytometry (FC) assessment of expression of GPI-anchored proteins (CD59 and CD55). Examining 51 blood samples from 48 patients, we found that the gel test is useful as a screening test for PNH diagnosis and can replace the Ham test and the sucrose lysis test. The threshold of the gel test is about 10 percent of defective RBCs detected by FC. It should, however, be supplemented with FC so as to analyze precisely the defective RBCs and granulocytes in patients with positive gel test results, and, in case of negative results, to detect a small clone of defective cells in atypical cases. Due to the simplicity of the gel test, its wide use can facilitate the diagnosis of PNH.     

Cloning of a CD59-like gene in rainbow trout. Expression and phylogenetic analysis of two isoforms

CD59, the major inhibitor of the complement membrane attack complex, is an 18-20 kDa glycoprotein, linked to the membrane via a glycosylphosphatidylinositol (GPI)-anchor. It restricts binding of C9 to the C5b-8 complex, preventing the formation of the complement membrane attack complex C5b-9. In this study we report the cloning of a second CD59-like gene in the rainbow trout, Oncorhynchus mykiss (referred to as CD59-2 and the previously deposited trout CD59-like gene as CD59-1). Trout CD59-2 is 56% identical to CD59-1 at the amino acid level. Both of trout CD59s show the highest identity score (54%) with putative CD59-like molecules from other teleost, and the overall identity with their mammalian orthologs is less than 30%. Trout CD59s are expressed in brain, heart, intestine, kidney, liver and spleen. Particularly, CD59-2 is abundant in trout brain, while CD59-1 seems to be absent in the trout spleen. Moreover, both of trout CD59 genes seems to be present as a single copy in trout genome.     

Latent infection of human herpesvirus 7 in CD4(+) T lymphocytes

To determine the cell populations in peripheral blood that are infected latently with human herpesvirus 7 (HHV-7), the real-time polymerase chain reaction (PCR) was used to determine the quantities of viral DNA in adherent and non-adherent cells from 71 healthy volunteers. Real-time PCR, which detected the U31 gene of HHV-7, was developed to measure viral load. The majority of non-adherent cells (14/16; 87.5%) contained HHV-7 DNA, while most of the adherent cells did not (1/16; 6.3%). HHV-7 viral load in non-adherent cells was significantly higher than that in adherent cells (P < 0.0001). Then, HHV-7 DNA load was compared between the CD4-positive and -negative cell fractions derived from the non-adherent cells of 26 healthy adults. As in the previous experiment, only 2 (7.7%) of the 26 adherent cell specimens contained small amounts of HHV-7 DNA (27.7 copies/1 x 10(6) cells and 208.7 copies/1 x 10(6) cells). In contrast, 88.5% of CD4(+) T cell samples (23/26 specimens) were positive for HHV-7 DNA, ranging from 0.4 to 3,542.8 copies/1 x 10(6) cells. Viral DNA was detected in only 3 (11.5%) of the 26 CD4(-) T cell specimens, with 8.4, 63.5, and 74.1 copies/1 x 10(6) cells. HHV-7-positive DNA loads were significantly higher in the CD4(+) T cells than those observed in the CD4(-) T cells (P = 0.0005). The relationship between HHV-7 viral loads in non-adherent cells and those in saliva was investigated. Comparison of HHV-7 DNA load between blood CD4(+) T cells and saliva revealed that the HHV-7 DNA load in saliva correlated with that present in CD4(+) T cells (r = 0.415; P = 0.0174).     

Applying the erythrocyte Pig‐a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation

The Pig‐a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)‐anchored cell surface markers. We hypothesized that the erythrocyte Pig‐a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI‐anchored CD59 as the Pig‐a mutation marker and examined the frequency of CD59‐negative sperm using flow cytometry. A reconstruction experiment that spiked un‐labeled sperm (mutant–mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant–mimic sperm, demonstrating the analytical ability for CD59‐negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N‐ethyl‐N‐nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid‐lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose‐dependent increase in the frequency of CD59‐negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59‐negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment‐related changes were detected in the frequency of CD59‐negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59‐negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig‐a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485–493, 2017. © 2017 Wiley Periodicals, Inc.     

A novel in vitro system to generate and study latently HIV-infected long-lived normal CD4+ T-lymphocytes

Studies of mechanisms of HIV-latency and its reactivation in long-lived resting CD4+ T-lymphocytes in patients have been limited due to the very low frequency of these cells ( approximately 1-10 cells per 10(6) CD4+ T-cells). To circumvent this obstacle, an in vitro culture system for post-activation long-term survival of normal CD4+ T-cells in a quiescent (non-cycling) state was developed and used to generate latently infected, long-lived quiescent CD4+ T-cells from HIV-infected, activated normal CD4+ T-lymphocytes. This yielded a frequency of approximately 5x10(4) latently infected cells per 10(6) cells in culture, which is approximately 10(3)- to 10(4)-fold higher than that available from patients. Moreover, 5-10% of long-term surviving non-cycling T-cells were found to make infectious HIV continuously at low levels, showing that HIV production from infected T-cells does not require full cellular activation. This model system should facilitate studies of long-lived, latently infected and persistently HIV-producing quiescent normal CD4+ T-lymphocytes.     

Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells.

The CAMPATH-1H (CD52) antigen is a 21 000-28 000 MW glycopeptide antigen that is highly expressed on T and B lymphocytes and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The humanized CAMPATH-1H anti-CD52 antibody is extremely effective at mediating depletion of both normal and tumorigenic lymphocytes in vivo and has been used in clinical trials for lymphoid malignancy and rheumatoid arthritis. Cross-linking GPI-anchored molecules, including CD52, on the surface of T lymphocytes in the presence of phorbol 12-myristate 13-acetate or anti-CD3, results in cellular activation. In the present study we have investigated the functional effects of cross-linking CD52 on T and B tumour cell lines. Cross-linking CD52 on either a B-cell line, Wien 133, which expresses high levels of endogenous CD52 or Jurkat T cells transfected and selected to express high levels of CD52 resulted in growth inhibition. This effect showed slower kinetics and occurred in a lower percentage of cells than growth inhibition stimulated via T- or B-cell receptors. Growth inhibition of the Wien 133 line was followed by the induction of apoptosis, which appeared independent of the Fas/Fas L pathway. Wien 133 cells surviving anti-CD52 treatment were selected and cloned and found to have down-regulated CD52 expression, with a characteristic biphasic pattern of 10% CD52-positive, 90% negative by fluorescence-activated cell sorter analysis. Interestingly, surface expression of other GPI-linked molecules, such as CD59 and CD55, was also down-regulated, but other transmembrane molecules such as surface IgM, CD19, CD20, HLA-DR were unaffected. The present study and previous work show that this is due to a defect in the synthesis of mature GPI precursors. Separation of CD52-positive and negative populations in vitro resulted in a rapid redistribution to the mixed population. Injection of CD52-negative cells into nude mice to form a subcutaneous tumour resulted in a substantial increase in expression of CD52. These results suggest that the defect in the Wien 133 cells is reversible, although the molecular mechanism is not clear. These observations have relevance to the clinical situation as a similar GPI-negative phenotype has been reported to occur in lymphocytes following CAMPATH-1H treatment in vivo.     

The effects of marathon running on expression of the complement regulatory proteins CD55 (DAF) and CD59 (MACIF) on red blood cells

Exercise is known to result in the haemolysis of red blood cells (RBCs). Although mechanical stressors such as footstrike and an increased velocity of blood flow may be involved, the biological mechanisms that underpin RBC haemolysis remain elusive. RBCs are potentially susceptible to lysis by autologous complement activation. RBCs are protected from the lytic effects of complement by regulatory proteins (CRPs) bound to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. This study aimed to determine if marathon running would result in RBC haemolysis through a loss of membrane expression of the CRPs CD55 (decay accelerating factor) and CD59 (membrane attack complex inhibitory factor). Blood samples were obtained from 14 male runners before, within 30 min after, and 24 h after completion of the 2004 London Marathon. RBCs were assessed for cell surface CD55 and CD59 expression using indirect immunofluorescence assays and flow cytometry. No significant changes in the total RBC count, haematocrit or haemoglobin concentrations were found in response to running the marathon (P > 0.05). Blood bilirubin concentrations after the marathon were significantly greater than the pre-race values (P < 0.01). The relative fluorescent intensity (arbitrary units) of CD55 and CD59 expression on RBC membranes did not change in response to the marathon race (P > 0.05). In conclusion, marathon running did not alter the expression of CD55 or CD59 on RBCs, despite concomitant elevations in blood bilirubin concentrations. Consequently, any haemolysis of RBCs that occurred in response to the marathon was not likely due to a loss of membrane bound CRPs and subsequent cell lysis by autologous complement.