Development of tertiary lymphoid structures in the kidneys of pigs with chronic leptospiral nephritis

Tertiary lymphoid organs (TLOs) are structures that are morphologically and functionally similar to secondary lymphoid organs. TLOs usually arise in a background of chronic inflammation. Several histological patterns of interstitial nephritis have been documented in porcine leptospirosis. Among them the lympho-follicular pattern is characterized by infiltrates of mononuclear cells organized in lymphoid follicle-like structures. Immunohistological analysis of 5 cases of porcine lympho-follicular nephritis associated with Leptospira Pomona infection demonstrated the presence of inflammatory cell populations, including B cells, T cells, macrophages and follicular dendritic cells (FDCs), which were compartmentalized as in TLOs. Immunohistochemistry for Leptospira Pomona revealed an intimate association between leptospiral antigen and FDCs. Overexpression of MHCII in different populations of both professional and non-professional antigen presenting cells was also demonstrated. FDCs play role during TLOs induction for their ability to retain non-self antigens in the form of immune complexes, thus causing persistent T cell activation, generation of a complex cytokine network and stimulation of humoral immunity. Sustained bacterial antigen presentation in the context of chronic leptospiral nephritis, may also lead to autoimmune mechanisms involved in the generation of TLOs. Whether lymphoid neogenesis and TLOs play a protective role in porcine leptospiral nephritis is still unclear.     

Features of follicular dendritic cells in ovine pharyngeal tonsil: an in vivo and in vitro study in the context of scrapie pathogenesis

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion "reservoir" during the latency phase, thus playing a key role during the scrapie lymphoinvasion.     

Age, scrapie status, PrP genotype and follicular dendritic cells in ovine ileal Peyer's patches

Follicular dendritic cells (FDCs) residing within ileal Peyer's patches (PPs) are of crucial relevance for sheep scrapie early pathogenesis and subsequent scrapie prion neuroinvasion. In this study, ileal PP follicles were significantly more numerous in lambs than in adult Sarda breed sheep, with significant differences being also found in lymphoid follicle area, perimeter and FDC density. Furthermore, PrPd deposition within ileal PPs and host's PrP genotype did not significantly influence these parameters. We conclude that age significantly affects FDC density in ileal PPs from Sarda breed ovines, independently from host's scrapie status and PrP genotype.     

CNA42 monoclonal antibody identifies FDC as PrPsc accumulating cells in the spleen of scrapie affected sheep

Natural scrapie, new variant Creutzfeldt-Jakob disease and murine experimental transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders. The agent responsible for these diseases is closely related to PrPsc, an abnormal isoform of the cellular prion protein. Before reaching the brain, it invades and replicates in lymphoid organs such as spleen, tonsils and lymph nodes. Follicular dendritic cells (FDC) may support the prion replication in lymphoid tissues of sheep as shown in murine models infected with scrapie. In sheep, specific antibodies recognising FDC are lacking. The CNA42 mAb, directed against human FDC was used to identify these cells in sheep spleen. As well as showing that the pre-treatments needed for immunohistochemical detection of PrPsc did not prevent labelling by the CNA42 mAb, accumulation of PrPsc in FDC of spleens of scrapie affected sheep was demonstrated using a double immunolabelling strategy. Thus, the CNA42 antibody represents a suitable tool to identify FDC and investigate their role in natural sheep scrapie.     

Antigen distribution in organs of mink with Aleutian disease parvovirus infection

On tissues from naturally infected non-Aleutian mink an immunohistological study was performed using monoclonal antibodies and the immunoperoxidase method. Structural proteins of ADV were demonstrated in cryosections and in ethanol-fixed and paraffin-embedded material which provide antigen detection in a similar amount together with good histological structure. In lymphoid organs viral antigen was restricted to B-cell areas, particularly lymphoid follicles. The pattern of antigen distribution was typical for follicular dendritic cells which are capable to retain immune complexes. Beside macrophages in the interior of lymphoid follicles most likely proliferating B-lymphoblasts reveal nuclear and cytoplasmatic presence of structural proteins indicating viral replication. Cells of the mononuclear phagocyte system such as cells of lymphatic sinuses and hepatic Kupffer cells harbor viral protein in the cytoplasm, probably resulting from phagocytosis of immune complexes. Renal glomeruli were consistently negative for virus antigen whereas in interstitial infiltrates cells resembling macrophages stained positive for ADV structural proteins.     

Characterization of NCR1+ cells residing in lymphoid tissues in the gut of lambs indicates that the majority are NK cells

Natural killer (NK) cells are important for immune protection of the gut mucosa. Previous studies have shown that under pathologic conditions NK cells, T cells and dendritic cells are found co-localised in secondary lymphoid organs where their interaction coordinates immune responses. However, in the gut-associated lymphoid tissues (GALTs), there are few detailed reports on the distribution of NK cells. Sheep harbour several types of organised lymphoid tissues in the gut that have different functions. The ileal Peyer’s patch (IPP) functions as a primary lymphoid tissue for B cell generation, while the jejunal Peyer’s patches (JPPs) and colon patches (CPs) are considered secondary lymphoid tissues. In the present study, we analysed tissues from healthy lambs by flow cytometry and in situ multicolour immunofluorescence, using recently described NCR1 antibodies to identify ovine NK cells. Most NCR1+ cells isolated from all tissues were negative for the pan T cell marker CD3, and thus comply with the general definition of NK cells. The majority of NCR1+ cells in blood as well as secondary lymphoid organs expressed CD16, but in the GALT around half of the NCR1+ cells were negative for CD16. A semi-quantitative morphometric study on tissue sections was used to compare the density of NK cells in four compartments of the IPPs, JPP and CPs. NCR1+ cells were found in all gut segments. Statistical analysis revealed significant differences between compartments of the primary lymphoid organ IPP and the secondary lymphoid organs of the JPPs and CP. NK cells co-localised and made close contact with T cells, dendritic cells and other NK cells, but did not show signs of proliferation. We conclude that NK cells are present in all investigated segments of the sheep gut, but that presence of other innate lymphoid cells expressing NCR1 cannot be excluded.     

Altered lymphocyte homeostasis after oral prion infection in mouse

Transmissible spongiform encephalopathies (TSEs) or prion diseases develop as central nervous system (CNS) disorders characterized by extremely long incubation periods. Although TSEs do not go along with inflammatory infiltrates and/or antibody production against the prion protein (PrP(Sc)), the immune system plays an important role in pathogenesis as long as different lymphoid organs (Peyer's patches, lymph nodes and spleen) may facilitate the accumulation and further spread of prions after peripheral exposure. In this work we investigated the changes in lymphoid and dendritic cell (DC) populations as well as the implications of different cytokines during disease progression after experimental oral inoculation of prions in a transgenic mouse model. At different days post-inoculation (dpi), T and B lymphocytes and DC populations from lymphoid organs, blood and brain were analyzed by flow cytometry and immunohistochemistry. Besides time related variations in lymphoid cell numbers due to the aging of the animals significant changes related with the infection were found in mesenteric lymph nodes, peripheral blood leukocytes (PBLs) as well as in spleen, affecting the CD4/CD8 ratio. In contrast, little or no variation was detected in Peyer's Patches or in thymus either associated with aging or the infection status. At individual time points significant differences between infected and control mice were seen in the CD8, CD4 and DC populations, with less evidence of differences in the B cell compartment. Finally, a pro-inflammatory phenotype occurred at early times in the spleen, where the levels of lymphotoxin-beta mRNA were found augmented with respect to controls. Altogether, these results suggest that normal regulation of lymphocyte populations becomes altered along the progression of a prion infection.     

Immmunohistochemical study of the blood and lymphatic vasculature and the innervation of mouse gut and gut-associated lymphoid tissue

The blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut-associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double-positive cells. Dendritic cells, the most important antigen-presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane-membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron-immune regulation/cross-talk and probably neuroinvasion of prion pathogens in the gut and GALT.     

Dendritic cells in cattle: phenotype and function

Dendritic cells are professional antigen presenting cells derived from the bone marrow and distributed throughout body tissues where they are located in sites that are suitable for antigen uptake. They are central to the induction of immune responses in naive animals and thus have become targets in strategies that are aimed at modulating resistance to infection. Studies in cattle have shown that the dendritic cells are phenotypically heterogeneous and that the different phenotypes have different biological properties. The molecular basis for this variation has begun to be investigated and has led to the identification of a member of the SIRPalpha family of signal regulatory proteins (MyD1) on a subset of dendritic cells in afferent lymph. Uptake of antigen by cattle dendritic cells is by a number of mechanisms that can involve endocytosis via clathrin coated pits or via caveolae as well as macropinocytosis.     

犬瘟热病毒自然感染犬淋巴组织中T、B细胞变化的研究

为了调查患犬瘟热病犬淋巴组织中T、B细胞变化的特点及淋巴细胞减少的发病机制,试验通过免疫组织化学的方法观察了T细胞(用CD3和CD45RO检测T细胞)、B细胞(用IgG、IgM抗血清检测B细胞)和犬瘟热病毒(抗犬瘟热病毒抗体)在病犬淋巴组织中的分布。结果表明:在淋巴组织中的淋巴细胞、淋巴小结中树突状细胞和巨噬细胞中均检出了抗病毒阳性反应细胞。在骨髓组织的前髓细胞中也发现抗病毒阳性反应细胞和嗜酸性胞浆内及核内包涵体的存在。与对照组相比,CD3和CD45RO阳性细胞主要存在于T细胞的分布域;但CD3和CD45RO阳性T细胞的数量较少。位于淋巴组织中的巨噬细胞有的被CD45RO染成阳性。在B细胞分布的区域中,IgG、IgM阳性细胞的数量明显减少;一些位于淋巴组织的浆细胞也被IgG或IgM染成阳性。在淋巴组织中淋巴细胞减少的顺序为:IgG阳性细胞减少最明显,其次为IgM和CD45RO阳性细胞,再次为CD3阳性细胞。依据试验结果,作者认为病犬淋巴组织中淋巴细胞减少主要是由B细胞缺乏所引起的;淋巴细胞的增殖能力减弱是引起淋巴组织中淋巴细胞减少的重要原因。     

Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens.

The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the right udder with HKS at 1 month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral (draining) and contralateral (non-draining) suprammammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen-specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory cell migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, immunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals.     

Pathogenesis and immunopathology of systemic and nervous canine distemper

Canine distemper is a worldwide occurring infectious disease of dogs, caused by a morbillivirus, closely related to measles and rinderpest virus. The natural host range comprises predominantly carnivores. Canine distemper virus (CDV), an enveloped, negative-sense RNA virus, infects different cell types, including epithelial, mesenchymal, neuroendocrine and hematopoietic cells of various organs and tissues. CDV infection of dogs is characterized by a systemic and/or nervous clinical course and viral persistence in selected organs including the central nervous system (CNS) and lymphoid tissue. Main manifestations include respiratory and gastrointestinal signs, immunosuppression and demyelinating leukoencephalomyelitis (DL). Impaired immune function, associated with depletion of lymphoid organs, consists of a viremia-associated loss of lymphocytes, especially of CD4+ T cells, due to lymphoid cell apoptosis in the early phase. After clearance of the virus from the peripheral blood an assumed diminished antigen presentation and altered lymphocyte maturation cause an ongoing immunosuppression despite repopulation of lymphoid organs. The early phase of DL is a sequel of a direct virus-mediated damage and infiltrating CD8+ cytotoxic T cells associated with an up-regulation of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α and IL-12 and a lacking response of immunomodulatory cytokines such as IL-10 and transforming growth factor (TGF)-β. A CD4+-mediated delayed type hypersensitivity and cytotoxic CD8+ T cells contribute to myelin loss in the chronic phase. Additionally, up-regulation of interferon-γ and IL-1 may occur in advanced lesions. Moreover, an altered balance between matrix metalloproteinases and their inhibitors seems to play a pivotal role for the pathogenesis of DL. Summarized, DL represents a biphasic disease process consisting of an initial direct virus-mediated process and immune-mediated plaque progression. Immunosuppression is due to early virus-mediated lymphocytolysis followed by still poorly understood mechanisms affecting antigen presentation and lymphocyte maturation.     

Immunocompetent cells of the turkey: age and organ distribution patterns of T and B lymphoid cells.

The percentage of lymphoid cells from the bursa of Fabricius, thymus, spleen, peripheral blood, and cecal tonsils reactin with chicken antisera to turkey bursa and thymus were evaluated, using 1-day-old to 5-week-old turkeys. For this, rabbit anti-chicken globulin fluorescein isothiocyanate conjugate was used. The percentage of lymphoid cells showing immunoglobulin surface determinants from these organs also was examined, using a direct immunofluorescence test with a rabbit anti-turkey globulin fluorescein isothiocyanate conjugate. This study suggests that the bursa-specific antigen and immunoglobulin surface determinants could be used as markers for bursa-derived cells in the turkey. It also was found that thymus-specific antigen could be used as a marker for thymus-derived cells.     

Immunohistochemical characterization of Kisselev nodules (ectopic lymphoid follicles) in wild boar (Sus scrofa L.)

This paper describes the histopathological features and cellular distribution of T lymphocytes (CD3), B cells (CD79), follicular dendritic cells (FDC) and macrophages (alpha-1-antitrypsin, lysozyme) in lymphoid aggregates (Kisselev nodules) found in the lung, kidney and liver of wild boar (Sus scrofa L.). The distribution of immunoreactive cells, tested for antibodies, was similar to that found in the cortex of lymph nodes: lymphoid follicles with germinal centers mainly consisting of CD79⁺ B cells with sparse interfollicular tissue (CD3⁺ T lymphocytes). This finding and the association of these structures with helminthic infections suggests that local humoral immunity is central to the organism’s response to parasitic challenge. The presence of follicular dendritic cells confirms the high degree of organization of these lymphoid-like structures. The role of other pathogenic factors and the induction of chronic inflammatory reaction in these ectopic lymphoid sites is also discussed.     

The effect of oral immunization on the population of lymphocytes migrating to the mammary gland of the sow

Sows were immunized orally with live Escherichia coli according to various immunization schedules. Six pregnant gilts were used; 4 immunized at various intervals during the last month of gestation, 1 control immunized after parturition following suppression of lactation by weaning and 1 non-immunized control. The effect of oral vaccination on cell populations from lymphoid organs was studied. The in vitro proliferative responses of the cell populations to K88 antigen, anti-Ig sera and mitogens were used to demonstrate the distribution of sensitized lymphocytes over different lymphoid organs. The capacity of these cells to produce antigen-specific Ig was determined by in ovo translation of their mRNA.Oral administration of antigen resulted in the appearance of K88-positive cells in lymphoid organs. In lactating sows, sensitized cells preferentially occured in the mammary lymph nodes, whereas after suppression of lactation such a distribution was not seen. A possible route of migration of sensitized lymphocytes is discussed in relation to the local immune response. The antibody isotype produced by sensitized lymphocytes seemed to depend on the immunization schedule. The most effective schedule was one starting early in gestation and comprising frequent administration of antigen. This caused an optimal distribution of sensitized lymphocytes capable of IgA production.     

Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue

To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720 h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from 72 h PI onwards. This work may be useful not only for offering a possibility of routine diagnosis of NGVE, but also for better understanding of the pathogenesis of the disease.     

New histochemical and ultrastructural observations on normal bovine tonsils.

Samples of normal bovine palatine tonsils were examined by light and electron microscopy. Like human tonsils, they were composed of crypts, subepithelial areas, follicles, and T-dependent zones, but their well-developed capsule subdivided the lymphoid tissue by connective septa. B cells formed the major lymphoid component. The follicles and T-dependent zones had morphological and histochemical features typical of peripheral lymph organs. Follicular dendritic cells were isolated and shown to be similar to human follicular dendritic cells.     

Oesophageal tonsil of the chicken

The oesophageal tonsil of the chicken is a novel member of the mucosal-associated lymphoid tissue (MALT), which is located around the entrance of the proventriculus. It consists of 6 to 8 single units, which are surrounded by a thin fibrous capsule. Each one is organised around the bottom of the longitudinal folds of the oesophagus, and serves as a 'tonsillar crypt'. Stratified squamous epithelium is infiltrated by lymphoid cells, i.e. T cells, plasma cells, macrophages, and dendritic cells, but not B cells, to form lymphoepithelium (LE). In the LE vimentin-, MHC II- and ATPase-positive cells possibly represent Langerhans' cells, but the appearance of 74.3 positive cells in the LE is unusual, because the 74.3 monoclonal antibody (mAb) recognises chicken follicular dendritic cells in the germinal centre and medulla of the bursal follicles. The subepithelial lymphoid tissue is organised into T- and B-dependent regions, which are the interfollicular areas and the germinal centres, respectively. Existence of high-endothelial venules in the interfollicular region suggests an extensive cellular connection between the oesophageal tonsil and the other lymphoid organs. In the resting oesophagus the lumen is closed, but during swallowing a bolus the crypt opens and the lymphoepithelium can be exposed to undigested food, antigens, infectious agents and vaccines. The location of the oesophageal tonsil, cranial to the stomach, may provide this organ with a unique role as compared to the other parts of the MALT; namely, it may contribute to the replication of infectious bursal disease virus (IBDV) and/or the pathogenesis of infectious bursal disease.     

Fusion of C3d molecule with bovine rotavirus VP7 or bovine herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immunization

The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization. One or two copies of murine C3d-cDNA, C3d or (C3d)(2), respectively, were cloned together with bovine rotavirus (BRV) VP7 or bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) genes. All constructs contained a signal peptide that resulted in the secretion of the expressed proteins. In vitro, the characterization of the chimeric proteins indicated that both VP7 and gD retained their antigenicity and the C3d remained biologically active. However, immunization with plasmids encoding VP7-C3d chimeras did not enhance rotavirus-specific antibody responses and the frequency of BRV-specific IFN-gamma secreting cells in the spleens were significantly lower in mice immunized with pVP7-(C3d)(2) when compared with mice immunized with plasmid encoding VP7. The same pattern of immune responses was observed for plasmids encoding gD-C3d. Both gD-specific antibody responses and the frequency of gD-specific IFN-gamma secreting cells were significantly lower in mice immunized with plasmid expressing gD-C3d chimeras when compared with mice immunized with plasmid encoding gD alone. These results indicate that co-expression of C3d with an antigen actually inhibit both humoral and cell-mediated antigen-specific immune responses.     

Multiorgan engraftment of human somatic cells in swine foetuses after intra-blastocyst transplantation

Adult human stem cells, mainly from hematopoietic lineage, have been injected into developing pre-immune animal foetuses, and xenogenic engraftment of liver and other organs has been reported. We isolated a rare cell population from adult human liver, fat and skin. Colonies with few cells became visible as early as 2-3 days, and a fully formed colony took 10-14 days to form. These colonies were named as liver-derived cell lines (LDCs), fat-derived cell lines (FDCs) and skin-derived cell lines (SDCs). All these cells express few pluripotency markers like Klf4, c-myc and Sox2. Pig blastocysts were injected with LDCs, FDCs and SDCs and transferred to recipient pigs. We achieved an overall pregnancy rate of 71.4% at day 35. The foetuses were analysed for human cell chimerism in liver, kidney and heart both by RT-PCR and real-time PCR using primers specific to human and pig mitochondrial DNA. The percentage of foetuses showing chimerism was 17.4% (4/23), 12.5% (2/16) and 11.1% (1/9) for LDCs, FDCs and SDCs, respectively. Of these, 42.9% (three out of seven) showed chimerism in liver and 71.4% (five out of seven) showed kidney chimerism. However, we did not detect any chimerism in the heart. The level of chimerism varied and was in the range of one human cell per one hundred thousand to one million pig cells.