Colonies Of Origin Research Articles

Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

Gene Expression in ESCs Derived From Cloned Blastocysts

Once harvested from the inner cell mass (ICM), embryonic stem cells (ESCs) undergo extended proliferation in culture as undifferentiated cells poised for multilineage differentiation. Confirmation of the pluripotent status of these cells involves both morphological criteria and specific molecular markers, and expression of typical marker genes. Nanog, a divergent homeodomain protein that directs propagation of undifferentiated cells, is down-regulated during early de-differentiation and becomes silent in completely differentiated cells. RNA from cloned and in vivo derived ESCs was extracted and qRT-PCR performed to assess their stemness. Molecular markers and morphological criteria were used to confirm ESC pluripotency. MII oocytes retrieved from B6D2-F1 mice were enucleated, injected with cumulus cell nuclei, and then activated using SrCl2. Blastocysts generated in vivo were similarly cultured and served as controls. Embryos were evaluated daily for their growth, hatching, and attachment to a feeder cell layer. After reaching an adequate size, ICMs were isolated mechanically 4 days later and trypsinized to provide single cells. RNA was isolated from ESCs derived from in vivo and cloned embryos, with mouse embryonic fibroblasts (MEF) serving as a negative control. qRT-PCR was performed with custom-designed primers for the target sequences of Nanog, Oct-4 (pluripotent markers), and Ttr genes, while Act-β and Gapdh were used as normalizers. Gene expression was reported as a proportion, calculated from the cycle threshold against Act-βconsidered at 100% expression. Quality and stemness were assessed according to morphological characteristics such as cell size, nucleus/cytoplasm ratio, and nucleolar patterns. In addition, pluripotency markers such as alkaline phosphatase activity, Oct-4 and TROMA-1 expression were also assessed. Of 17 oocytes that survived cloning, 12 constructs displayed two pronuclei, 4 developed further to produce fully expanded blastocysts, and 2 produced ESC lines of good quality. On the other hand, 40 (87.5%) in vivo-derived zygotes developed into blastocysts, and once plated 7 (20.0%) produced ESC lines. ESC colonies of both origin contained at least 60% of undifferentiated cells according to morphological characteristics and molecular markers. Between 10,000 and 30,000 cells processed generated 0.3 to 0.9 μg of total RNA that displayed two ribosomal subunits. Reverse transcription and qRT-PCR were successful in all specimens. As expected, Nanog was not expressed in MEFs whereas the in vivo and cloned-derived ESC cells revealed a similar 74.6% and 66.6% expression, respectively (χ2 = 1.19). Similarly, Oct-4 was not expressed in MEFs while the in vivo and cloned-derived ESC had an upregulation of 76.9% and 67.8%. MEFs manifested a higher expression (69.6%; P = 0.001) of Ttr (a marker of differentiation) compared to the in vivo and cloned-derived ESC colonies (40.7 and 49.2%, respectively). Results coming from a genomic indicator accord with those from other molecular markers in establishing stemness of the ESC colonies. Microarray analysis showed that clone-derived ESCs have similar expression patterns to those generated in vivo, and therefore that they may be used for stem cell therapy.

Length of life, age at first foraging and foraging life of Africanized and European honey bee (Apis mellifera) workers, during conditions of resource abundance

SUMMARYThis study was conducted to analyse the length of life, the age at which bees began to forage, and the foraging life of European and Africanized worker honey bees (Apis mellifera) during conditions of resource abundance. Newly emerged worker bees were identified with coloured paint or numbered plastic tags on their thorax, and were co-fostered in common colonies of European, Africanized or unrelated bee origin to infer if the variation found was due to genetic or environmental effects. Both European and Africanized bees varied significantly in their length of life, within a range of 19.9 to 25.9 days, but there was no clear and consistent difference for this trait between the two types of bees. Hive and colony environment influenced the length of life of both bee types. Bees lived longer in colonies with Africanized environment. These results suggest that genetic effects influence the length of life of workers bees to a lesser degree than do environmental effects. The age at which bees began foraging affected both the length of life and the foraging life of the experimental workers. Bees that started to forage late in life lived longer than those that started early. Additionally, bees that initiated foraging activities at a younger age had a longer foraging life. The strongest correlation was between length of life and foraging life (ρ = 0.66), indicating that bees that lived longer also had a longer foraging life. Bees with the longest foraging lives were those that began to forage early in life and that also lived as long or longer than average bees. European bees had longer foraging lives than Africanized bees. The implications of these results on the life-history strategies of Africanized and European bees are discussed.

Evaluation of the defensive behavior of two honeybee ecotypes using a laboratory test

Honeybee defensive behavior is a useful selection criterion, especially in areas with Africanized honeybees (Apis mellifera L). In all genetic improvement programs the selected characters must be measured with precision, and because of this we evaluated a metabolic method for testing honeybee defensive behavior in the laboratory for its usefulness in distinguishing between honeybee ecotypes and selecting honeybees based on their level of defensive responses. Ten honeybee colonies were used, five having been produced by feral queens from a subtropical region supposedly colonized by Africanized honeybees and five by queens from a temperate region apparently colonized by European honeybees. We evaluate honeybee defensive behavior using a metabolic test based on oxygen consumption after stimulation with an alarm pheromone, measuring the time to the first response, time to maximum oxygen consumption, duration of activity, oxygen consumption at first response, maximum oxygen consumption and total oxygen consumption, colonies being ranked according to the values obtained for each variable. Significant (p < 0.05) differences were detected between ecotypes for each variable but for all variables the highest rankings were obtained for colonies of subtropical origin, which had faster and more intense responses. All variables were highly associated (p < 0.05). Total oxygen consumption was the best indicator of metabolic activity for defensive behavior because it combined oxygen consumption and the length of the response. This laboratory method may be useful for evaluating the defensive behavior of honey bees in genetic programs designed to select less defensive bees.

Partial Seasonal Isolation of African and European-Derived &lt;I&gt;Apis mellifera&lt;/I&gt; (Hymenoptera: Apidae) Drones at Congregation Areas from Subtropical Mexico

The genetic interactions between European and African-derived honey bees in the Neotropics are unclear. To study the abundance of males of each type, two apiaries with colonies of African-derived honey bee and European honey bee origin were established in the surroundings of one drone congregation area. The frequencies of African-derived honey bee and European honey bee males in the mating area were studied by taking samples of drones at the drone congregation area twice a month for 5 mo. Drones from each type of colony were identified by the use of two allozyme polymorphic systems: malate dehydrogenase (Mdh), hexokinase (Hk), and by haplotype using the EcoRI site of the mitochondrial DNA. The results of a Fisher exact test showed that, although Mdh alleles had similar frequencies across the time of study (P = 0.095), the frequency of Hk alleles and haplotypes in drones varied between months (P = 0.0001). Early in March, the frequencies of African-derived honey bee-typical alleles were significantly higher compared with European honey bee-typical alleles. However, at the end of the season of drone production in July, European honey bee-typical alleles were significantly more frequent in drones than those of the African-derived honey bee type. The results mirror the findings of an early peak of drone brood production reported for African-derived honey bee colonies compared with a peak later in the year in European honey bee ones. It is concluded that the seasonal frequencies of African-derived honey bee and European honey bee drones in the mating areas are not static. They vary in accordance with the different peaks of male production in their respective colonies. This behavior may act as a partial genetic barrier between bee types. The implications of these findings with respect to current levels of Africanization in this region of Mexico and for queen rearing in Africanized areas are discussed.

Salivary proteins and glycoproteins in phlebotomine sandflies of various species, sex and age.

Salivary gland proteins were studied in sandflies (Diptera: Psychodidae: Phlebotominae) by electrophoretic techniques. In Phlebotomus duboscqi Neveu-Lemaire the protein concentration was about 30 times higher in females than in males. SDS PAGE revealed eight major bands of 29-62 kDa in salivary gland extracts (SGE) from females, whereas only one band of 57kDa was detected in males. The number of protein components in SGE gradually increased with the age of females. In P. papatasi (Scopoli) the typical electrophoretic pattern was reached in 3-5 days after imago emergence, depending on the temperature at which females were maintained. All major protein components of the female SGE were present in the content of glands. Female SGE were compared in seven colonies of five Phlebotomus and Lutzomyia species; electrophoretic profiles distinguished between species and even between colonies of different geographical origin. In general, the highest variability of major protein components was observed in the 38-48kDa region. Four colonies of the subgenus Phlebotomus (P. duboscqi and P. papatasi) possessed common mobility polypeptides, the highest similarity was found between two colonies of P. papatasi. Other species tested significantly differed, specific prominent bands of 33, 35 and 38kDa were found in P. halepensis Theodor, P. perniciosus Newstead and Lutzomyia longipalpis (Lutz & Neiva), respectively. Glycoproteins in SGE of Lu. longipalpis and P. duboscqi females were identified and analysed using blotting with five lectin conjugates. Specific reaction of lectins ConA and WGA revealed the complex type of N-glycans in the 48 and 53-54kDa glycoproteins present in both species. Similar glycosylation was detected in species-specific bands of the 57-60 and 65-67 kDa in P. duboscqi and Lu. longipalpis, respectively. The high mannose type of glycosylation was found in the 20 and 39 kDa polypeptides of Lu. longipalpis and the 40-42 kDa polypeptides of P. duboscqi. Innate lectin activity specific for aminosugars was detected in SGE of P. duboscqi females using haemagglutination tests with rabbit erythrocytes.

Host F1 Mice Pretreated With Granulocyte Colony-Stimulating Factor Accept Parental Bone Marrow Grafts in Hybrid Resistance System

AbstractIn the setting of hybrid resistance, parental C57BL/6 bone marrow (BM) grafts are vigorously rejected by lethally irradiated (C57BL/6xDBA/2) F1 mice. However, F1 mice pretreated by continuous administration of granulocyte colony-stimulating factor (G-CSF ) with a miniosmotic pump before BM grafting developed day-8 splenic colonies of donor origin. This inhibitory effect on rejection was reversible because F1 mice regained the capacity to reject parental BM when the pump ceased functioning. The appearance of only a small number of colonies with the administration of G-CSF soon after BM grafting suggested the importance in producing this inhibitory effect of pre-exposure of host mice to G-CSF. Because G-CSF administration with a syngeneic combination did not influence the number of colonies, an altered distribution of grafted precursors was unlikely. The absence of a reduction in the number of NK1.1-positive cells in G-CSF–treated mice suggested functional impairment of natural killer cells, major effectors in hybrid resistance, but further study is necessary to elucidate the mechanism underlying this phenomenon. However, our results indicate the importance of G-CSF as a regulator in a certain type of immune response and raise the possibility of clinical application in transplantation medicine.

The taxonomic identity of the cosmopolitan prymnesiophyte Phaeocystis: a morphological and ecophysiological approach

Phaeocystis species diversity has been reviewed by comparing the morphological and physiological characteristics of Phaeocystis cells and colonies of different geographical origin. These analyses gave evidence for four Phaeocystis species: P. globosa, P. scrobiculata, P. pouchetii and one undefined antarctic species, distinguishing themselves by colony and single cell morphology and temperature tolerance. Typical colonial shape constitutes the most apparent morphological characteristics distinguishing P. pouchetii from P. globosa. Differences between colonies referable to pouchetii and globosa can be confirmed on the basis of variation in temperature and light requirements, as well as morphological descriptions of palmelloid stages, e.g. colony shape and size, organisation of the cells inside the colonies. The most striking features of the motile single cell are the thread-like appendages, which are much longer than the cell itself, the organic scales covering the cells, varying in shape and size, the haptonema and the flagella. On this basis, previous Phaeocystis records were analysed and the geographical distribution of the genus reported. There was no evidence for strain specific elemental composition or photosynthesis or growth performance of cells and colonies. This indicates that more elaborate molecular and biochemical analyses are required to identify different species. Possible opportunities available through modern chemical and molecular biological advances are described.

Detection of DNA polymorphisms in predatory coccinellids using polymerase chain reaction and arbitrary primers (RAPD-PCR)

DNA polymorphisms were identified in some Coccinellid predators that are being tested as biological control agents against aphids and other insects in North America. The technique employs a variation of the Polymerase Chain Reaction (PCR), called RAPD-PCR, that uses single arbitrarily selected primers to amplify a random group of genomic sequences. Using this technique it was possible to distinguish among laboratory reared colonies of diverse geographic origin. Several colonies each, of three species were examined (Coccinella septempunctata, Hippodamia variegata, andPropylea quatuordecimpunctata). It was also possible to distinguishC. septempunctata from a closely related speciesC. transversoguttata biinterrupta. The technique promises to be a very useful source of markers for maintaining colonies and tracking genes in biological control projects and in identifying species and immature stages of insects.

Highly efficient elimination of Philadelphia leukemic cells by exposure to bcr/abl antisense oligodeoxynucleotides combined with mafosfamide.

Synthetic oligodeoxynucleotides complementary to the break-point junction of bcr-abl transcripts selectively inhibit the proliferation of Philadelphia-positive leukemic cells, but residual leukemic cells persist in antisense oligodeoxynucleotides-treated cultures. Cyclophosphamide derivatives such as mafosfamide and 4-hydroperoxycyclophosphamide are used at high doses for purging of Philadelphia leukemic cells from marrows but such treatment can be associated with delayed engraftment and prolonged cytopenias. To develop a more effective procedure that might optimize the killing of leukemia cells and the sparing of normal hematopoietic progenitor cells, a 1:1 mixture of Philadelphia leukemic cells and normal bone marrow cells was exposed to a combination of a low dose of mafosfamide and bcr-abl antisense oligodeoxynucleotides and assayed for growth ability in clonogenic assays and in immunodeficient mice. Bcr-abl transcripts were not detected in residual colonies, and cytogenetic analysis of individual colonies revealed a normal karyotype. Normal but not leukemic hematopoietic colonies of human origin were also detected in marrows of immunodeficient mice 1 mo after injection of the treated cells. Our results indicate that a combination of a conventional chemotherapeutic agent and a tumor-specific antisense oligodeoxynucleotide is highly effective in killing leukemic cells and in sparing a much higher number of normal progenitor cells as compared with high-dose mafosfamide treatment. This offers the prospect of a novel and more selective ex vivo treatment of chronic myelogenous leukemia.

The Phytopathological Significance of Mycelial Individualism

Phytopathologists have long been aware of the considerable intraspecific variation that can exist in natural populations of phytopathogenic mycelial fungi. However, the significance of this variation has largely been perceived in terms of fungal-plant rather than interfungal relationships. Corresponding­ ly, questions about variation in the pathogenicity and virulence of different fungal lineages have received more attention than questions about the way in which these lineages have the potential to interact antagonistically or syn­ ergistically with one another (55). The purpose of this article is to explore the many ways in which mycelial interactions may, nonetheless, be relevant to our understanding of the etiology and control of plant disease. An important step in understanding mycelial interactions is to have a clear perspective of the units of selection in natural fungal populations. Historical­ ly, this issue has been clouded both by the indeterminate nature of fungal mycelia, which have the potential to continue extension indefinitely, and by the concept that hyphal anastomosis could allow extensive cooperation be­ tween colonies of diverse genetic origin (22, 57, 58). The resultant view that members of the same species could pool their genetic resources in a versatile

Vegetative reproduction in the octocoral Briareumasbestinum (Pallas)

Briareum asbestinum (Pallas), a common octocoral of Caribbean reefs, produces runner-like branches which are capable of attaching to the substratum and establishing new colonies. The subsequent death of the intervening runner leaves groups of genetically identical but physiologically distinct colonies. The origin of these colonies can be deduced by the presence or absence of runners. Analysis of colonies at Marsarkantupo in the San Blas Islands, Panama and at Carrie Bow Cay, Belize indicate that 23–56% and 18–67%, respectively, of the colonies at these sites originate from vegetative runners. In both areas the distribution of colonies of larval origin is not uniform. Colonies originating from larvae are patchily distributed within habitats and are over-represented in shallow habitats. The ability of B. asbestinum to reproduce vegetatively results in the spread and development of large populations of this species in many habitats where it might otherwise be rare.

A method for obtaining high quality chromosome preparations from single hemopoietic colonies on a routine basis

The need for improved methodology to facilitate cytogenetic analysis of hemopoietic stem cell populations, particularly in studies of the hemopoietic malignancies, has been recognized for a number of years. Since primitive hemopoietic cells can be stimulated to form colonies of identifiable progeny under appropriate conditions in vitro, it should in theory, be possible to obtain such information. However, hemopoietic colonies of human origin rarely contain more than 1000 cells, and in handling such small samples, cell loss has historically been a major problem. We now describe a method that has allowed from two up to more than 50 metaphases per colony to be obtained from most erythroid and granulopoietic colonies harvested individually from standard methylcellulose assay cultures. Of key importance is the selection of large but still immature colonies and the use of polylysine coated slides, which ensures recovery of 80–90% of the sample. The method yields reproducibly high quality metaphases suitable for analysis after G, Q, and fluorescent-reverse banding. Cytogenetic analysis of 58 colonies removed individually from cultures initiated with a mixture of male and female cells showed both male and female colonies to be present as expected. In all instances only one type of metaphase was ever found in a single colony. A procedure for measuring the incidence of sister chromatid exchange (SCE) in the progeny of primitive hemopoietic progenitors has also been established. SCE values obtained for 1- to 2-week-old colonies derived from normal progenitors were similar to previously published normal SCE values. Preliminary analysis of erythroid and granulocytic colonies from one patient with polycythemia vera (PV) and two patients with chronic myelogenous leukemia (CML) indicate that preparations of equal quality are obtained from such individuals and thus, for the first time, the Ph 1 chromosome could be readily demonstrated in erythroid colonies. Further application of this method to the investigation of patients with myeloproliferative disease is now underway.

Cellular regulation of fetal hemoglobin synthesis in man: Investigation of γ and β mRNA accumulation in clonal erythroid cultures initiated from erythroid progenitors derived from fetuses, neonates, and adult individuals

The accumulation of globin mRNA in erythroid cells during human development was investigated in clonal cultures of fetal, newborn, and adult origin erythroid progenitors and in control uncultured erythroblasts and reticulocytes. The relative concentrations of α, β, and γ mRNA sequences were measured by hybridization with purified α, β, and γ[ 3H]cDNA probes. We found that the amount of γ mRNA is characteristic of the developmental stage from which the erythroid progenitor cells are obtained. In colonies from fetal liver cells, 89% of the non-α mRNA is γ mRNA. Colonies derived from neonatal (cord blood) cells produce an average of 55% γ mRNA, while colonies generated from either adult bone marrow or progenitors circulating in the peripheral blood of the adult individual produce γ mRNA ranging from 15 to 38% of the non-α sequences. When compared with erythroid cells (erythroblasts or reticulocytes) from the same tissues, the fetal liver colonies produce identical proportions of γ mRNA as liver cells in vivo. Colonies from circulating erythroid progenitors from the perinatal (switchover) period accumulate somewhat less γ mRNA (55%) than do circulating reticulocytes in vivo (64%). The concentration of γ mRNA (15 to 38%) in colonies of adult marrow or peripheral blood origin is substantially higher than the trace amounts (<1%) found in mature erythroid cells in vivo. The amounts of β and γ globin chain synthesis from fetal liver, cord blood, and adult peripheral blood are proportional to β and γ mRNA ( r = 0.97) and thus indicate that selective translation of β and γ mRNA is not a significant factor in the switch from Hb F to Hb A production. (However, both neonatal reticulocytes in vivo and erythroid colonies in vitro contain excess non-α mRNA but exhibit balanced globin chain synthesis; translational control mechanisms apparently prevent excess synthesis of total non-α chains.) The expression of stage-specific patterns of γ and β mRNA in erythroid colonies suggests that the erythroid progenitor cells in vivo possess the potential for unique programs of γ and β mRNA accumulation before they actually enter the pathway to terminal maturation.

The seasonal life cycle ofAnoplolepis longipes (Jerdon) (Hymenoptera: Formicidae) in a cacao plantation and under brushed rain forest in the northern district of Papua New Guinea

The only period of sexual brood production during the year occurred at the beginning of the wet season. Dispersal of mature sexuals from their colonies of origin took place in the third and fourth months of the wet season. However, dispersal of alate females was not apparent from nests at the cacao site. The worker caste was produced throughout the wet season and reached a peak after the dispersal of mature sexual castes. For the rest of the year they were produced at a reduced level, dependent in part upon the harshness of the dry season.

The detection of monosomic colonies produced by mitotic chromosome non-disjunction in the yeast Saccharomyces cerevisiae

A diploid yeast strain, D6, is described which monitors mitotic non-disjunction by the phenotypic expression of a set of coupled and recessive markers flanking the centromere of chromosome VII. These markers are not expressed in the heterozygous condition prevailing in D6. The left arm of chromosome VII carries a tightly centromere linked marker, leu1 (leucine requirement), distal to leu1 in this order: trp5 (trytophan requirement), cyh2 (recessive resistance to cycloheximide) and met 13 (requirement for methionine). The right arm is marked with ade3 (simultaneous requirement for adenine and histidine). D6 is homozygous for ade2 and consequently, forms red rather than the normally white colonies. It shows no requirement for the above amino acids and it is sensitive to cycloheximide. Unmasking of all the markers on chromosome VII leads to colonies that are white because ade3 sets a block preceding the ade2 block (which causes the accumulation of a precursor of the red pigment), they require leucine, tryptophan and methionine, and grow on media with cycloheximide. Cells are plated on a cycloheximide medium where red and white colonies are formed. Colonies of spontaneous origin were tested. The majority of the white colonies expressed all the recessive markers whereas only few of the red colonies expressed all the markers on the left arm of chromosome VII. Basically expression of recessive markers on both sides of the centromere can be explained as a result of two coincident events of mitotic crossing over. However, the frequency of colonies expressing centromere linked leu1 was 14 times higher among the white types than the red ones. This suggested that the white, cycloheximide resistant, leucine requiring colonies arose by mitotic non-disjunction and not only by two coincident mitotic crossing over events. Presumptive spontaneous monosomic segregants were placed on sporulation medium. Only 8 out of 30 isolates sporulated, which showed that these eight segregants were diploid at the time of sporulation. They could have arisen by two coincident crossover events or through restoration of a normal disomic condition after non-disjunction had occurred. The genetic data thus leaves us with only its statistical argument in favour of non-disjunction. Further confirmation of monosomic nature of the white cycloheximide resistant colonies was provided by the estimates of their DNA contents. Compared to the stock wild type diploids the presumptive monosomics showed a reduction in DNA content. We have utilized D6 to investigate the possible induction of mitotic non-disjunction after treatment with gamma rays, heat shock at 52°C and ultraviolet irradiation. In all cases white, cycloheximide resistant colonies were produced at levels significantly higher than that found in untreated cultures. In order to detect the production of monosomic cells, treated cultures were grown for 48 h in non-selective medium after exposure to allow for “expression” of the monosomic condition.

Sex hormones and the regulation of erythroid spleen colonies development of fetal liver origin

The development of erythroid colonies of fetal liver hemopoietic cell origin in adult irradiated polycythemic mice was studied. It was found that orchidectomy sharply reduced the number of erythroid colonies developed in the spleen of these polycythemic male recipients. Estrogen injection to the orchidectomized polycythemic recipient did not further decrease the number of erythroid colonies developed. It is concluded that the development of erythroid colonies of fetal liver origin in polycythemic male recipients is maintained mainly by testicular testosterone. The complete suppression of these colonies in female recipients does not seem to be a result of inhibition by estrogen.

Sex Hormones and the Regulation of Erythroid Spleen Colonies Development of Fetal Liver Origin

The development of erythroid colonies of fetal liver hemopoietic cell origin in adult irradiated polycythemic mice was studied. It was found that orchidectomy sharply reduced the number of erythroid colonies developed in the spleen of these polycythemic male recipients. Estrogen injection to the orchidectomized polycythemic recipient did not further decrease the number of erythroid colonies developed. It is concluded that the development of erythroid colonies of fetal liver origin in polycythemic male recipients is maintained mainly by testicular testosterone. The complete suppression of these colonies in female recipients does not seem to be a result of inhibition by estrogen.

Antigens Induced By The Mouse Leukemia Viruses

Publisher Summary The chapter provides an overview of the immunology of mouse leukemia. Leukemia-inducing agents have been found in a variety of normal and malignant mouse tissues, thus, indicating their widespread distribution among mouse colonies of different origin. By using electron-microscopic techniques, the localization in the tissues and cells of infected and leukemic animals as well as the morphological structure of the leukemia viruses has been thoroughly investigated. The chapter also discusses various antigens induced by the mouse leukemia viruses and the tests that are available to diagnose them. Some of these tests include immunofluorescence tests (indirect immunofluorescence test) and cytotoxic tests (chromium-51 technique, fluorochromatic test, and microscopic evaluations). Currently, a number of leukemia-specific antigens detected by different methods are known to develop after virus infection. Not only are there antigens in leukemia cells detected by transplantation studies or by cytotoxic and fluorescent antibody techniques, but there are also antigens that are not restricted to leukemia cells such as soluble antigens demonstrable by adsorption to indicator cells, by immunoprecipitation, and by complement fixation. Immunological tolerance to virus-induced surface antigens is apparently an important precondition for the outgrowth and spreading of cells transformed to malignancy by leukemia viruses. Immunological tolerance to virus-induced antigens is a promoting factor in the leukemogenesis induced by certain viruses. Tolerance also favors the outgrowth and generalization of malignant cells.

Hemopoietic Spleen Colony Studies

Summary and Conclusions Suspensions of lymph node or thymus cells from normal mice, administered intravenously to lethally irradiated (820 to 950 r) isogenic mice in doses of 1 to 60 × 106 (viable) cells, were found to have very few spleen-colony-forming units (CFU). These were detectable only if high cell doses were administered to sufficiently large numbers of irradiated mice. Phytohemagglutinin (PHA) treatment of recipients appeared to increase slightly the frequency of spleen colonies in mice injected with thymus cells, and sometimes in mice injected with lymph node cells. Unpublished data indicate that this effect may be attributed, at least in part, to PHA stimulation of endogenous spleen colony formation. PHA treatment of donor mice slightly increased the low frequency of spleen colonies induced by lymph node cells, but not of colonies induced by thymus cells. PHA treatment of both donor and recipient had no additional effect on the numbers of spleen colonies formed by transfusion of thymus or lymph node cells. Histologically, cytologically and cytochemically the colonies formed by injection of thymus and lymph node cells were indistinguishable from the several types of hemopoietic spleen colonies induced in much larger numbers by bone marrow cell transfusions, with the following exception. They occurred against a background of significant diffuse lymphocytic repopulation of the spleen and partial repopulation of the lymphoid follicles caused by the large doses of lymphoid cells necessary to induce macroscopic spleen colonies, and contained lymphocytes of that background population. No macroscopic lymphopoietic colonies were found. Erythroid spleen colonies induced by transfusion of thymus or lymph node cells (the only type of spleen colony that might be misdiagnosed as “lymphoid colonies” on routine histologic examination) were found to take up 59Fe, to stain for hemoglobin and to be repressed by polycythemia. This repression was prevented by administered erythropoietin. These findings are identical to those obtained with erythropoietic colonies induced by bone marrow cells. On retransplantation, the spleen colonies induced by lymph node cells were shown to give the same spectrum of “clean” hemopoietic secondary spleen colonies as those induced by spleen colonies of bone marrow origin. There was no background of, or admixture with, diffuse lymphocytic repopulation of the spleen. Significant numbers of hemolytic antibody-forming cells were found in the spleen colonies induced by lymph node cells from PHA-treated donors when the irradiated recipients had also received injections of sheep red blood cells (SRBC). However, the concentration of antibody-forming cells in the colonies was only about one-fourth that in the intercolonial tissue. Spleen colonies induced by bone marrow injection did not contain antibody-forming cells, but comparable numbers of antibody-forming cells were produced in all of the hemopoietic colonies induced by bone marrow cells, and in the intercolonial tissue, by injecting lymph node cells from normal mice along with the bone marrow cells. Irradiated mice injected with 5 to 50 × 106 lymph node cells from normal donor mice, and with SRBC, had few or no spleen colonies, but sheep hemolytic antibody-forming cells appeared in their spleens in direct proportion to the number of lymph node cells administered. It is concluded that the spleen colonies induced by thymus or lymph node cells, with or without PHA-treatment of the donor or irradiated recipient, are not “lymphoid clones.” Rather, they are hemopoietic spleen colonies similar to those induced by bone marrow cells, with the exception that they are contaminated by the large numbers of transfused lymph node cells necessary to induce a few hemopoietic colonies. As such, they are unsuitable for clonal lymphoid studies for which they have been used and recommended.