Stable Lineage Research Articles

Occurrence of a Pig Respiratory Disease Associated with Swine Influenza A (H1N2) Virus in Tochigi Prefecture, Japan

In February 2008, a feeder pig herd of the affected farm in Tochigi Prefecture, Japan, showed increasing respiratory symptoms; by April, the situation worsened with 12-16 pigs dying daily. Diagnostic tests revealed the presence of H1N2 subtype of swine influenza virus (SIV) and Pasteurella multocida from nasal swab and lung emulsion. Serological tests by hemagglutination inhibition method and enzyme-linked immunosorbent assay method (ELISA; imported from U.S.A.) indicated the spread of SIV into the pig herds of the affected farm around April 2008. The severe infection and subsequent damage were considered as a result of the combined infection of SIV (H1N2) and bacteria that may have been prevalent in the pig farm. Genetic homology search of sequences for the hemagglutinin (HA) and neuraminidase (NA) genes of A/swine/Tochigi/1/08 showed high homology to Japanese SIVs (H1N2) isolated in the 2000s. Therefore, we considered that Japanese SIV (H1N2) has established an independent stable lineage and participated in infecting pig populations as one of the factors of the pig respiratory disease complex. Consistent surveillance would contribute to clarifying the prevalence of dominant SIVs.

Immune modulation: Turncoat regulatory T cells

Immune modulation: Turncoat regulatory T cells

Trypanosoma cruzi IIc: Phylogenetic and Phylogeographic Insights from Sequence and Microsatellite Analysis and Potential Impact on Emergent Chagas Disease

Trypanosoma cruzi, the etiological agent of Chagas disease, is highly genetically diverse. Numerous lines of evidence point to the existence of six stable genetic lineages or DTUs: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe. Molecular dating suggests that T. cruzi is likely to have been an endemic infection of neotropical mammalian fauna for many millions of years. Here we have applied a panel of 49 polymorphic microsatellite markers developed from the online T. cruzi genome to document genetic diversity among 53 isolates belonging to TcIIc, a lineage so far recorded almost exclusively in silvatic transmission cycles but increasingly a potential source of human infection. These data are complemented by parallel analysis of sequence variation in a fragment of the glucose-6-phosphate isomerase gene. New isolates confirm that TcIIc is associated with terrestrial transmission cycles and armadillo reservoir hosts, and demonstrate that TcIIc is far more widespread than previously thought, with a distribution at least from Western Venezuela to the Argentine Chaco. We show that TcIIc is truly a discrete T. cruzi lineage, that it could have an ancient origin and that diversity occurs within the terrestrial niche independently of the host species. We also show that spatial structure among TcIIc isolates from its principal host, the armadillo Dasypus novemcinctus, is greater than that among TcI from Didelphis spp. opossums and link this observation to differences in ecology of their respective niches. Homozygosity in TcIIc populations and some linkage indices indicate the possibility of recombination but cannot yet be effectively discriminated from a high genome-wide frequency of gene conversion. Finally, we suggest that the derived TcIIc population genetic data have a vital role in determining the origin of the epidemiologically important hybrid lineages TcIId and TcIIe.

Differential Virological and Immunological Outcome of Severe Acute Respiratory Syndrome Coronavirus Infection in Susceptible and Resistant Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2

We previously reported that transgenic (Tg) mice expressing human angiotensin-converting enzyme 2 (hACE2), the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), were highly susceptible to SARS-CoV infection, which resulted in the development of disease of various severity and even death in some lineages. In this study, we further characterized and compared the pathogeneses of SARS-CoV infection in two of the most stable Tg lineages, AC70 and AC22, representing those susceptible and resistant to the lethal SARS-CoV infection, respectively. The kinetics of virus replication and the inflammatory responses within the lungs and brains, as well as the clinical and pathological outcomes, were assessed in each lineage. In addition, we generated information on lymphocyte subsets and mitogen-mediated proliferation of splenocytes. We found that while both lineages were permissive to SARS-CoV infection, causing elevated secretion of many inflammatory mediators within the lungs and brains, viral infection appeared to be more intense in AC70 than in AC22 mice, especially in the brain. Moreover, such infection was accompanied by a more profound immune suppression in the former, as evidenced by the extensive loss of T cells, compromised responses to concanavalin A stimulation, and absence of inflammatory infiltrates within the brain. We also found that CD8(+) T cells were partially effective in attenuating the pathogenesis of SARS-CoV infection in lethality-resistant AC22 mice. Collectively, our data revealed a more intense viral infection and immunosuppression in AC70 mice than in AC22 mice, thereby providing us with an immunopathogenic basis for the fatal outcome of SARS-CoV infection in the AC70 mice.

Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage?

Regulatory T (T(Reg)) cells constitute a unique T-cell lineage that has a crucial role in immunological tolerance. Several years ago, forkhead box P3 (FOXP3) was identified as the transcription factor that was responsible for determining the development and function of these cells. However, the underlying mechanisms that are involved in the regulation of the FOXP3 gene remain unclear and therefore preclude accurate identification and manipulation of T(Reg) cells. In this Progress article, we summarize recent advances in understanding how FOXP3 expression is controlled and highlight evidence suggesting that epigenetic regulation of the FOXP3 locus contributes to its role as a lineage-specification factor.

A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

Th memory for interleukin‐17 expression is stable in vivo

Based on the memory for the re-expression of certain cytokine genes, different subsets of Th cells have been defined. In Th type 1 (Th1) and Th2 memory lymphocytes, the genes for the cytokines interferon-gamma and interleukin (IL)-4 are imprinted for expression upon restimulation by the expression of the transcription factors T-bet and GATA-3, respectively, and epigenetic modification of the cytokine genes. In Th17 cells, IL-17 expression is dependent on the transcription factors RORgammat and RORalpha. Here, we analyze the stability and plasticity of IL-17 memory in Th17 cells. We have developed a cytometric IL-17 secretion assay for the isolation of viable Th cells secreting IL-17. For Th17 cells generated in vitro, IL-17 expression itself is dependent on continued TGF-beta/IL-6 or IL-23 signaling and is blocked by interferon-gamma and IL-4 signaling. In response to IL-12 and IL-4, in vitro generated Th17 cells are converted into Th1 or Th2 cells, respectively. Th17 cells isolated ex vivo, however, maintain their IL-17 memory upon subsequent in vitro culture, even in the absence of IL-23. Their cytokine memory is not regulated by IL-12 or IL-4. Th17 cells generated in vivo are a stable and distinct lineage of Th cell differentiation.

Sociality, selection, and survival: Simulated evolution of mortality with intergenerational transfers and food sharing

Why do humans survive so long past reproductive age, and why does juvenile mortality decline after birth, both contrary to the classic theory of aging? Previous work has shown formally that intergenerational transfers can explain both these patterns. Here, simulations confirm those results under weaker assumptions and explore how different social arrangements shape life-history evolution. Simulated single-sex hunter-gatherers survive, forage, reproduce, and share food with kin and nonkin in ways guided by the ethnographic literature. Natural selection acts on probabilistically occurring deleterious mutations. Neither stable population age distributions nor homogeneous genetic lineages are assumed. When food is shared only within kin groups, an infant death permits reallocation of its unneeded food to the infant's kin, offsetting the fitness cost of the death and weakening the force of selection against infant mortality. Thus, evolved infant mortality is relatively high, more so in larger kin groups. Food sharing with nonkin reduces the costs to kin of child rearing, but also reduces the resources recaptured by kin after an infant death, so evolved infant mortality is lower. Postreproductive adults transfer food to descendants, enhancing their growth and survival, so postreproductive survival is selected. The force of selection for old-age survival depends in complicated ways on the food-sharing arrangements. Population-level food sharing with nonkin leads to the classic pattern of constant low mortality up to sexual maturity and no postreproductive survival.

DNA methylation controls Foxp3 gene expression

Compelling evidence suggests that Foxp3-expressing CD25(+)CD4(+) regulatory T cells (Treg) are generated within the thymus as a separate lineage. However, Foxp3(+)CD4(+) Treg can also be generated de novo in a TGF-beta-dependent process from naive T cells by TCR triggering. Recently, we have shown that naturally occurring, but not in vitro TGF-beta-induced Foxp3(+) Treg display stable Foxp3 expression that was associated with selective demethylation of an evolutionarily conserved element within the Foxp3 locus named TSDR (Treg-specific demethylated region). Here, we report that inhibition of DNA methylation by azacytidine, even in absence of exogenous TGF-beta, not only promoted de novo induction of Foxp3 expression during priming, but also conferred stability of Foxp3 expression upon restimulation. Most notably, such stable Foxp3 expression was found only for cells displaying enhanced TSDR demethylation. In contrast, in vitro TSDR methylation diminished its transcriptional activity. Foxp3(+) Treg generated in vivo by DEC-205-mediated targeting of agonist ligands to dendritic cells showed long-term survival in the absence of the inducing antigen and exhibited efficient TSDR demethylation. Together, our data suggest that TSDR is an important methylation-sensitive element regulating Foxp3 expression and demonstrate that epigenetic imprinting in this region is critical for establishment of a stable Treg lineage.

T Cells are Capable of Effector and Regulatory Function Simultaneously during Activation (88.3)

Abstract CD4+CD25+ regulatory T (Treg) cells are an important arm of the immune system that downregulate potentially harmful effector immune responses. FOXP3 expression and in vitro suppressor function have been considered to be the hallmarks of a unique and stable lineage of T-cells with immune regulatory function. Here we show that human CD4+CD25−FOXP3− T-cells attain a transient FOXP3+CD25+ state following activation. In this state of activation, these cells possess the classic marker profile and suppressive phenotype of regulatory/suppressor T-cells. However, the vast majority of these cells eventually down regulate FOXP3 and suppressor activity. Cytokine studies indicate that they are also capable of secreting effector cytokines when they are regulatory in function. Interestingly, CD8+CD25−FOXP3− T-cells also exhibit similar dynamics following activation. Our results show that, in humans, FOXP3 expression and Treg functionality are not distinct features of a separate lineage of T-cells, but rather a state attained by almost all T-cells during their activation. This suggests that attempts to increase “Tregs” in humans may paradoxically result in increased effector T-cells, with potentially deleterious effects. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts. This work was supported by grants (to N.J.K) from the NIH and the National MS society NMSS.

Phylogenetic Analysis of Low-Pathogenicity Avian Influenza H6N2 Viruses from Chicken Outbreaks (2001–2005) Suggest That They Are Reassortants of Historic Ostrich Low-Pathogenicity Avian Influenza H9N2 and H6N8 Viruses

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically isolated from South African ostriches, but during 2002 the first recorded outbreak of LPAI (H6N2) in South African chickens occurred on commercial farms in the Camperdown area of KwaZulu/Natal (KZN) Province. Sequence analysis of all eight genes were performed and phylogenetic analysis was done based on the hemagglutinin and neuraminidasc sequences. Results from phylogenetic analyses indicated that the H6N2 chicken viruses most likely arose from a reassortment between two South African LPAI ostrich isolates: an H9N2 virus isolated in 1995 and an H6N8 virus isolated in 1998. Two cocirculating sublineages of H6N2 viruses were detected, both sharing a recent common ancestor. One of these sublineages was restricted to the KZN province. The neuraminidase gene contained a 22-amino acid deletion in the NA-stalk region, which is associated with adaptation to growth in chickens, whereas the other group, although lacking the NA-stalk deletion, spread to commercial farms in other provinces. The persistence of particular H6N2 types in some regions for at least 2 yr supports reports from Asia and southern California suggesting that H6N2 viruses can form stable lineages in chickens. It is probable that the ostrich H6N8 and H9N2 progenitors of the chicken H6N2 viruses were introduced to ostriches by wild birds. Ostriches, in which AI infections are often subclinical, may serve as mixing vessels for LPAI strains that occasionally spill over into other poultry.

Epigenetic Control of the foxp3 Locus in Regulatory T Cells

Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4+ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3+CD25+CD4+ Tregs, but not in naïve CD25−CD4+ T cells. Partial DNA demethylation is already found within developing Foxp3+ thymocytes; however, Tregs induced by TGF-β in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-β–induced Foxp3+ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses

The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) A/Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased N-glycosylation, and a D144 mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens.

Persistence, Transmission, and Virulence Characteristics of Aeromonas Strains in a Duckweed Aquaculture-Based Hospital Sewage Water Recycling Plant in Bangladesh

The persistence and transmission of Aeromonas in a duckweed aquaculture-based hospital sewage water treatment plant in Bangladesh was studied. A total of 670 samples from different sites of the hospital sewage water treatment plant, from feces of hospitalized children suffering from diarrhea, from environmental control ponds, and from feces of healthy humans were collected over a period of three years. In total, 1,315 presumptive Aeromonas isolates were biochemically typed by the PhenePlate rapid screening system (PhP-AE). A selection of 90 representative isolates was further analyzed with PhenePlate (PhP) extended typing (PhP-48), fatty acid methyl ester analysis, and amplified fragment length polymorphism (AFLP) fingerprinting. In addition, the prevalence of the putative virulence factors hemolysin and cytotoxin and the presence of the cytolytic enterotoxin gene (AHCYTOEN) were analyzed. Aeromonas was found at all sites of the treatment plant, in 40% of the samples from environmental control ponds, in 8.5% of the samples from hospitalized children suffering from diarrhea, and in 3.5% of samples from healthy humans. A significantly high number of Aeromonas bacteria was found in duckweed, which indicates that duckweed may serve as a reservoir for these bacteria. PhP-AE typing allowed identification of more than 192 distinct PhP types, of which 18 major PhP types (MTs) were found in multiple sites and during several occasions. AFLP fingerprinting revealed the prevalence of genotypically indistinguishable Aeromonas isolates among certain PhP MTs recovered from different sampling occasions and/or at multiple sites. Hemolytic and cytotoxic activities were observed in 43% of the tested strains, whereas 29% possessed the cytolytic enterotoxin gene AHCYTOEN. Collectively, two specific MTs associated with diarrhea were shown to exhibit high cytotoxicity. Furthermore, all tested isolates of these major types were positive for the cytolytic enterotoxin gene. In conclusion, our data indicate that certain phenotypically and genotypically stable clonal lineages of Aeromonas have persisted in the treatment system for a prolonged period and might spread from the hospitalized children suffering from diarrhea to fish produced for human consumption through the sewage water treatment system.

Transient regulatory T-cells: A state attained by all activated human T-cells

CD4 +CD25 +FOXP3 + regulatory T-cells (T regs) form an important arm of the immune system responsible for suppressing untoward immune responses. T regs can be thymically derived or peripherally induced, even from CD4 +CD25 −FOXP3 − T-cells. FOXP3 expression and in vitro suppressive activity are considered unique hallmarks of this dedicated and stable lineage of regulatory cells. Here we show that virtually all human CD4 +CD25 −FOXP3 − T-cells and CD8 +CD25 −FOXP3 − T-cells attain a transient FOXP3 +CD25 + state during activation. In this state of activation, these cells possess the classic phenotype of T regs, in that they express similar markers and inhibit in vitro proliferation of autologous CD4 +CD25 − T-cells. This state is characterized by suppressed IFN-γ production and robust TNF-α and IL-10 production. Interestingly, the great majority of the activated cells eventually downregulate FOXP3 expression, with a concomitant drop in suppressive ability. Our results show that, in humans, FOXP3 expression and T reg functionality are not exclusive features of a stable or unique lineage of T-cells but may also be a transient state attained by almost all T-cells. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts and suggest that attempts to induce “T regs” may paradoxically result in induction of effector T-cells, unless stability is confirmed.

Artifical transfer and morphological description of virus particles associated with superparasitism behaviour in a parasitoid wasp

In parasitoids, the adaptive significance of superparasitism (laying of egg(s) in already parasitized hosts) has been the subject of strong controversy. The current view is to interpret this behaviour as an adaptation to increased competition for hosts, because the supernumerary egg still has a chance to win possession for the host. However, we recently discovered that in the solitary parasitoid Leptopilina boulardi, superparasitism is rather caused by an unknown infectious element: stable non superparasitizing lineages (NS) are transformed into stable superparasitizing lineages (S) after eggs from both lineages have competed inside the same host (superparasitism). In this report, we investigate the nature and location of the causative agent. Involvement of bacteria is unlikely because antibiotic treatments do not affect wasp phenotype and because bacterial 16S ribosomal DNA was not detected using PCR. We report successful injection experiments showing that the causative agents are located in wasp poison gland and ovaries and are stably inherited. Electron microscopic studies demonstrate that long filamentous virus particles located in wasp oviducts are strongly associated with superparasitism behaviour, leading to reconsider the adaptive significance of this behaviour in parasitoids. Interestingly, parasitoids are often infected with similar viruses for which no phenotypic effect has been documented. This raises the possibility that they could induce the same behavioural manipulation.

Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: Role of the BMP-4 gene

Previous studies showed that exposure of C3H10T1/2 stem cells to bone morphogenetic protein-4 (BMP-4) produced cells that convert into adipocytes at high frequency when treated with differentiation inducers. In the present investigation, an independent approach shows that BMP-4 is required for stable commitment of pluripotent stem cells to the adipocyte lineage. Exposure of proliferating 10T1/2 stem cells to 5-azacytidine, a potent DNA methylation inhibitor, gave rise to a subpopulation of cells that can be cloned and that have the capacity to undergo conversion into adipocytes upon treatment with terminal differentiation inducers. Detailed studies performed with a cloned committed subline, the A33 line, verified stable adipocyte lineage determination in the absence of exogenous BMP-4. Remarkably, this cell line expresses and secretes BMP-4 during proliferation in the same time window that exogenous BMP-4 must be added to naïve 10T1/2 cells to induce maximal adipocyte commitment. Furthermore, exposure of A33 cells to noggin, a naturally occurring BMP-4-binding antagonist, during this critical time window blocks subsequent differentiation. The role of BMP-4 in adipocyte lineage commitment is further strengthened by gene expression profiling of proliferating 10T1/2 stem cells and A33 preadipocytes. These findings revealed changes in the molecular circuitry, specifically coordinated changes in the expression of members of the BMP-4 signaling pathway, that distinguish A33 preadipocytes from uncommitted parental 10T1/2 stem cells. Together, these studies provide compelling evidence for the participation of BMP-4 in adipocyte lineage determination.

Chryseobacterium piscium sp. nov., isolated from fish of the South Atlantic Ocean off South Africa

Four isolates from freshly caught fish samples obtained from the South Atlantic Ocean off the South African coastline were shown to represent a novel species in the genus Chryseobacterium by means of a polyphasic taxonomic study. The four isolates had virtually identical whole-cell protein profiles, fatty acid profiles and biochemical properties. Analysis of the 16S rRNA sequence of strain LMG 23089(T) revealed 99.3 and 98.9 % similarity to the 16S rRNA sequences of the type strains of Chryseobacterium balustinum and Chryseobacterium scophthalmum, respectively. Strain LMG 23089(T) and the C. balustinum and C. scophthalmum type strains formed a stable lineage supported by a bootstrap value of 100 %. The levels of DNA-DNA hybridization towards these nearest phylogenetic neighbours were below 57 %. The absence of growth on MacConkey agar or at 37 degrees C (on nutrient agar), the capacity to grow in the presence of 5 % NaCl and the production of urease activity differentiate this novel taxon from C. balustinum and C. scophthalmum. The four isolates are formally classified as Chryseobacterium piscium sp. nov., with strain LMG 23089(T) (=CCUG 51923(T)) as the type strain. Its DNA G + C content is 33.6 mol%.

Initial T cell frequency dictates memory CD8+ T cell lineage commitment.

Memory T cells can be divided into central memory T cell (T(CM) cell) and effector memory T cell (T(EM) cell) subsets based on homing characteristics and effector functions. Whether T(EM) and T(CM) cells represent interconnected or distinct lineages is unclear, although the present paradigm suggests that T(EM) and T(CM) cells follow a linear differentiation pathway from naive T cells to effector T cells to T(EM) cells to T(CM) cells. We show here that naive T cell precursor frequency profoundly influenced the pathway along which CD8+ memory T cells developed. At low precursor frequency, those T(EM) cells generated represented a stable cell lineage that failed to further differentiate into T(CM) cells. These findings do not adhere to the present dogma regarding memory T cell generation and provide a means for identifying factors controlling memory T cell lineage commitment.

327 CHARACTERIZATION OF FETAL AND ADULT FIBROBLASTS FROM NELORE BOVINE FOR NUCLEAR TRANSFER

Cloning by nuclear transfer is a technology that has provided major advances in reproductive sciences. It is an efficient method: (1) to produce transgenic embryos, fetuses, and animals; (2) to multiply genetically superior adult animals; and (3) to generate proteins, tissues, and organs for xenotransplants of human interest. Basic studies on nuclear transfer have contributed to understanding how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies. The aim of this study was to establish a stable lineage of Nelore adult and fetal fibroblasts for embryo reconstruction. Cultures were characterized by morphological aspects, and by immunocytochemical and ultrastructural analysis. Cultures of fetal and adult fibroblasts at passage 2 stained positively for cytokeratin, a typical protein from the intermediate filament of epithelial cells, in some cells but staining was not found in subsequent passages. A dense and organized network occurred in adult and fetal fibroblast cultures presenting positive immunostainig for vimentin in pass 5, 10, and 15 subcultures, but did not stain for cytokeratin (negative control). The results demonstrated that fibroblasts continued expressing vimentin and maintained their morphological aspects in culture. The ultrastructural analysis showed the presence of organelles involved in protein synthesis including dilated rough endoplasmatic reticulum, Golgi cisternae, and polyribosomes. Elongate mitochondria were distributed around the Golgi complex, suggesting high metabolic activity. These results demonstrated that adult and fetal fibroblasts continued to develop biological activities and maintained specific characteristics under these culture conditions. In conclusion, adults and fetal fibroblasts can be successfully used as donor cells for nuclear transfer. This work was supported by FAPESP 01/11931-8 and 01/13944-0.