Artificial Construction of Immune Tissues/Organoids and Their Application for Immunological Intervention.

Human-type lymphoid tissue organoids, which stably function in our body for a certain period of time or longer, may have a great potential as immune-stimulatory or immune-regulatory devices and could be utilized in the future for the treatment of various diseases such as cancer, severe infection, autoimmunity and congenital as well as acquired immunodeficiency resulting from severe infections or aging. In this review, we discuss about rationality and trials of the synthesis of immunologically functional lymphoid tissue organoids mainly in mouse. We have been recently trying to construct immunologically functioning human-type organoids, and the efforts are also briefly described.

Laparoscopic Free Omental Lymphatic Flap for the Treatment of Lymphedema.

UNLABELLED: Advances in microsurgery have displayed promising results for the treatment of lymphedema. The use of vascularized lymph node transfers has increased in popularity but incurs the potential risk for donor-site lymphedema. The omentum has been previously described for the treatment of lymphedema but has been overlooked because of presumed high morbidity, including the need for celiotomy and pedicled complications. The authors present a novel technique and early results of the laparoscopic free omental lymphatic flap for the management of lymphedema. The minimally invasive harvest successfully avoids both the previously associated morbidity of this flap and the risk of iatrogenic lymphedema to the donor site. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Human hemato-lymphoid system mice: current use and future potential for medicine.

To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.

Cutting edge: lymphoid tissue inducer cells maintain memory CD4 T cells within secondary lymphoid tissue.

Phylogeny shows that CD4 T cell memory and lymph nodes coevolved in placental mammals. In ontogeny, retinoic acid orphan receptor (ROR)γ-dependent lymphoid tissue inducer (LTi) cells program the development of mammalian lymph nodes. In this study, we show that although primary CD4 T cell expansion is normal in RORγ-deficient mice, the persistence of memory CD4 T cells is RORγ-dependent. Furthermore, using bone marrow chimeric mice we demonstrate that LTi cells are the key RORγ-expressing cell type sufficient for memory CD4 T cell survival in the absence of persistent Ag. This effect was specific for CD4 T cells, as memory CD8 T cells survived equally well in the presence or absence of LTi cells. These data demonstrate a novel role for LTi cells, archetypal members of the innate lymphoid cell family, in supporting memory CD4 T cell survival in vivo.

Lymphatic tissue transplant in lymphedema--a minimally invasive, outpatient, surgical method: a 10-year follow-up pilot study.

Lymphedema is mainly characterized by swelling, fibrosis, and non-pitting edema. The aim of this study was evaluation of the long-term (10 years) effects of autologous lymphatic tissue implant in lymphedema. Lymphatic tissue from 9 patients (harvested form the same patient in areas not affected by lymphedema) was reimplanted into the affected limb, and these patients were followed for 10 years. Lymph nodes were harvested at the neck, axillary, or inguinal space (contralateral limb). Results showed that limb volume was decreased in the treatment group vs. controls. In ultrasound, black, low density, lymphatic spaces were visible in 100% of patients at inclusion but in only 23% of these subjects at 10 years. Thus, this early report proposes a new, minimally invasive method to improve lymphedema. Studies in progress will indicate the role of lymphatic transplant in the management of lymphedema and the best indications for this method.

Requirements for follicular exclusion and competitive elimination of autoantigen-binding B cells.

Results from several mouse tolerance models indicate that autoreactive B cells in peripheral lymphoid organs develop an anergic phenotype, migrate to the boundary between the T cell zone and the B cell follicle (T/B boundary), and undergo rapid cell death. We have used B cells from mice that are double-transgenic for soluble hen egg lysozyme (HEL) and an Ig that recognizes HEL with a high affinity to characterize the mechanisms underlying the migration and elimination of autoreactive B cells. In contrast to the situation for acutely activated B cells, we find that anergic B cells have reduced levels of CXCR5, the receptor for the follicular chemokine, CXCL13, and this contributes to their exclusion from follicles. CCR7 expression is required for follicular exclusion of anergic cells, although up-regulation of the receptor does not appear to be necessary. By TUNEL analysis, we observe that excluded anergic cells die in situ at the T/B boundary. We also show that this elimination occurs via a Fas-independent mechanism. Using CCR7(-/-)Ig(HEL)-transgenic B cells we find that localization to the T/B boundary is not a necessary event to achieve the competitive elimination of autoantigen-binding B cells. These findings characterize the mechanism for follicular exclusion of autoantigen-binding B cells and they indicate that B cells compete for survival by mechanisms that are separate from competition for the follicular niche.

[Microsurgical transplantation of "lymph flaps"]. / Mikrokhirurgicheskaia transplantatsiia "limfaticheskikh loskutov".

The review is concerned with the main historical stages in the development of "lymph flaps" transplantation. The author describes their blood supply, the topography of the constituent lymph structures, the feeding vascular pedicle, techniques of the harvesting, revascularization and restoration of lymph drainage. The review is intended for familiarization of a wide circle of physicians with the current strategy of microsurgical treatment of extremity lymphedema. This will allow to widen the outlook of practicing physicians and will give a possibility of further perfection of this trend.

Distribution of cycling T lymphocytes in blood and lymphoid organs during immune responses.

Proliferation of murine T lymphocytes in blood, lymph nodes, and spleen was studied in four in vivo stimulation systems, using BrdU pulse-labeling of DNA-synthesizing cells. The T cell response to the superantigen Staphylococcus enterotoxin B (SEB) was studied in detail. Vbeta8+ T cells showed a peak of DNA synthesis 16-24 h after SEB injection, and the percentage of BrdU+ CD4 and CD8 T cells was higher in blood than in lymph nodes and spleen. DNA synthesis was preceded by massive migration of Vbeta8+ cells from blood to lymphoid organs, in which the early activation marker CD69 was first up-regulated. SEB-nonspecific Vbeta6+ cells showed minimal stimulation but, when cycling, also expressed a high level of CD69. The other systems studied were injection of the IFN-gamma inducer polyinosinic:polycytidylic acid, infection by the BM5 variants of murine leukemia virus (the causative agent of murine AIDS), and T cell expansion after transfer of normal bone marrow and lymph node cells into recombinase-activating gene-2-deficient mice. In each case, a peak of T cell proliferation was observed in blood. These data demonstrate the extensive redistribution of cycling T cells in the first few hours after activation. Kinetic studies of blood lymphocyte status appear crucial for understanding primary immune responses because cycling and redistributing T lymphocytes are enriched in the circulating compartment.

Follicular dendritic cell (FDC) precursors in primary lymphoid tissues.

The origin of follicular dendritic cells (FDC) is unresolved, and as such, remains controversial. Based on the migration of Ag-transporting cells (ATC) into lymphoid follicles and the phenotypic similarity between FDC and ATC, one hypothesis is that ATC may represent emigrating FDC precursors. This contrasts with the view that FDC originate from local stromal cells in the secondary lymphoid tissues. Mice homozygous for the severe combined immunodeficiency (prkdc(scid)) mutation (scid) lack FDC. Thus, they provide a powerful tool for assessing de novo generation of FDC. To test whether FDC precursors could be found in bone marrow or fetal liver, scid/scid mice were reconstituted with either: 1) bone marrow cells from (BALB/c x C57BL/6)F1 donors, 2) bone marrow cells from ROSA BL/6 F1 (lacZ-transfected) mice, 3) rat bone marrow cells, or 4) rat fetal liver cells. Six to eight weeks after reconstitution with F1 bone marrow, cells reactive with the FDC-labeling mAb, FDC-M1, also expressed donor class I molecules on their surfaces. Similarly in mice reconstituted with lacZ-transfected bone marrow cells, these cells were also positive for the lacZ gene product. Furthermore, in spleens of animals reconstituted with either rat bone marrow or rat fetal liver, rat FDC were identified using the specifically labeling mAb, ED5. In all cases, host FDC were also present, indicating that scid/scid mice have FDC precursors that will mature in the presence of allogeneic or xenogeneic lymphoid cells. In summary, FDC can be derived from progenitor cells present in primary lymphoid tissues.

Both resting and activated B lymphocytes expressing engineered peptide-Ig molecules serve as highly efficient tolerogenic vehicles in immunocompetent adult recipients.

To test the potential for genetically transferring foreign sequences into autologous cells for specific modulation of immunity, we have generated transgenic mice that express an engineered peptide-IgG construct in the peripheral B cell compartment. B cells from these mice express and can be stimulated to secrete a murine IgG1 chain grafted with residues 12-26 from bacteriophage A cI repressor protein in-frame at the heavy chain N terminus. As expected, 12-26-IgG transgenic mice are profoundly tolerant to the peptide at both the T and B cell levels. Importantly, the injection of transgenic whole spleen, purified B cells, or even bone marrow cells into normal, immunocompetent adults results in profound peptide-specific T cell tolerance, as well as partial B cell tolerance. Injection of LPS-activated peptide-Ig-expressing B cells was uniquely effective at diminishing an ongoing humoral immune response typical of both Th1 and Th2 help. Since fixed transgenic B cells were tolerogenic, this suggests that secretion of the fusion protein is not required for tolerogenicity. These results show that an engineered self Ig, as well as B lymphocytes expressing epitopes from such a fusion protein, can regulate both cellular and humoral immune responses. Moreover, these studies provide the basis for expressing foreign epitopes on engineered IgG for the induction of gene-transferred tolerogenesis in autoimmune states.

IL-12 p40 messenger RNA expression in target organs during acute graft-versus-host disease. Possible involvement of IFN-gamma.

The onset of acute graft-vs-host disease (aGVHD) is accompanied by macrophage (M phi) priming and the presence of bacteria-derived LPS in the sera of transplanted animals. Priming of M phi occurs during aGVHD despite the suppression of T cell function. We have investigated whether IL-12 mediates the continued production of IFN-gamma during the state of T cell immunosuppression that accompanies aGVHD. Acute GVHD was induced in nonirradiated AxC57BL/6F1 mice by the injection of C57BL/6 lymphoid cells. Despite T cell immunosuppression, M phi became primed, as shown by their expression of inducible nitric oxide synthase mRNA and their production of nitric oxide in response to LPS. Continual exposure to IFN-gamma was required to maintain a primed state in M phi during aGVHD. IL-12 p40 peptide mRNA was increased in M phi purified from animals undergoing aGVHD 14 days after transplantation. Target organs of aGVHD, including thymus, salivary gland, and lung, showed increased IFN-gamma mRNA between days 7 and 14 after transplantation. The increase was accompanied by an induction of mRNA for the p40 peptide of IL-12 and inducible nitric oxide synthase within the target organs. These results provide evidence for localized production of IFN-gamma within aGVHD target organs and suggest that it is mediated by LPS-induced production of IL-12 by M phi. Our data elucidate the mechanism of activation of M phi during aGVHD that results in TNF-alpha and nitric oxide production and delineates the effector role of M phi in the pathology of aGVHD.

Experience of a new surgical procedure for the treatment of unilateral obstructive lymphedema of the lower extremity: adipo-lymphatico venous transfer.

We report five cases of adipo-lymphatico venous transfer (ALVT) performed to treat unilateral obstructive lymphedema of the lower extremity. ALVT is a surgical procedure that utilizes the long saphenous vein and its surrounding lymphatic tissue from the unaffected limb for the treatment of lymphedema. Since ALVT does not necessitate anastomoses of lymphatic vessels, it can be performed regardless of the severity and duration of lymphedema, and stable long-term results can be obtained when the transferred lymphatic tissue shows viability.

Reduced provirus burden and enhanced humoral immune function in AZT-treated SCID-feline mice inoculated with feline immunodeficiency virus.

The lack of a safe, economical murine lentivirus model for human immunodeficiency virus type 1 (HIV-1) infection of humans has hampered the preclinical evaluation of potential antiviral compounds, vaccines, and biological response modifiers. A small animal model that does not employ HIV-1 is needed to minimize risk of accidental human exposure, enhance efficient use of scarce experimental compounds, and reduce laboratory space necessary to conduct statistically significant in vivo trials. Feline immunodeficiency virus (FIV), an immunosuppressive lentivirus of domestic cats, has been used extensively as an animal model for the pathogenesis and therapy of human HIV-1 infection. Cats, however, are not amenable to large-scale efficacy trials because of their relatively large size, high cost, and limited degree of physiologic characterization, particularly with regard to drug metabolism. To adapt the feline immune system to a small laboratory animal host, severe combined immunodeficient mice (SCID mice) were engrafted with feline lymphoid tissues (forming the SCID-fe mouse) and inoculated with FIV. Two quantitative parameters, the incidence of provirus detection in feline tissue grafts and the level of feline IgG in plasma, were used to demonstrate the antiviral efficacy of 3'-azido-3'-deoxythymidine (AZT, azidothymidine, Retrovir, zidovudine) in the SCID-fe system. Of 17 SCID-fe mice inoculated with 7 x 10(6) peripheral blood mononuclear cells (PBMC) from an FIV-infected cat, eight had detectable FIV provirus in both the feline thymus and feline lymph node implants, as measured by polymerase chain reaction (PCR)/Southern blot analysis. Treatment of these mice with AZT at a dose of 125 mg kg-1 day-1 in drinking water beginning 1 day prior to FIV inoculation and continuing throughout the study interval prevented the dual detection of provirus in feline lymph node and thymus grafts of all mice tested. In a separate experiment, the level of spontaneous feline IgG production was quantified by ELISA 2 weeks after FIV inoculation with and without AZT treatment. Mean plasma feline IgG level of five SCID-fe mice inoculated with 10(3) TCID50 cell-free FIV was 2.23 mg ml-1. Mean feline IgG level of five mice inoculated with the same quantity of FIV and treated with AZT beginning 1 day prior to virus inoculation and continuing for 2 weeks thereafter was 14.98 mg ml-1. AZT significantly (P < 0.05) enhanced feline humoral immune function at a virus inoculum titer of 10(3) TCID50.(ABSTRACT TRUNCATED AT 400 WORDS)

Dose-dependent systemic human immunodeficiency virus infection of SCID-hu mice after intraperitoneal virus injection.

SCID mice were engrafted with human foetal liver, thymus and lung. Human cells were subsequently detected among peripheral blood leukocytes for 81% of tested animals and in tissue implants for 100% of tested animals. SCID-hu mice received intraperitoneal injections of human immunodeficiency virus type 1 (HIV1) at from 20 up to 20,000 median tissue culture infectious doses (TCID5). HIV1 infection was detected by means of cell culture and polymerase chain reaction both in blood and implants, up to 58 days after infection. The rate of infection was dependent upon the inoculated dose: the frequency of thymus infection ranged from 14% with 20-500 TCID50 up to 100% with 20,000 TCID50. HIV1 infection was detected less frequently in blood leukocytes than in thymus. Thymus virus load ranged from 40 to 50,000 HIV1 provirus copies per million cells and was not correlated with either infectious dose or viraemia. Thymus T-cell depletion was observed mainly in the CD1+4+8+ immature thymocyte compartment. The same rate of SCID-hu mouse infection was obtained using three different primary HIV1 isolates, suggesting that infection was not restricted to a few particular virus strains. The systemic infection of SCID-hu mice following intraperitoneal virus injection mimics some traits of human HIV infection and provides a promising, novel approach for future investigations in this field.

Transplantation of T cell-mediated, lymphoreticular disease from the scurfy (sf) mouse.

The X-linked mutation, scurfy (sf), causes a fatal lymphoreticular disease characterized by runting, lymphadenopathy, splenomegaly, hypergammaglobulinemia, exfoliative dermatitis, Coombs'-positive anemia, and death by 24 days of age. T lymphocytes are required to mediate this syndrome as shown by a total absence of disease in mice bred to be scurfy and nude (sf/Y; nu/nu). The scurfy phenotype is not transmitted by sf/Y bone marrow transplants, though cells of scurfy origin do reconstitute all lymphoid organs in the recipient mouse. These data suggest that scurfy disease results from an abnormal T cell development process and not from an intrinsic stem cell defect. We therefore tested the ability of transplanted scurfy thymuses to transmit scurfy disease to congenic euthymic mice, to athymic (nude) mice, and to severe combined immunodeficiency (SCID) mice. Euthymic recipients of sf/Y thymic grafts remained clinically normal as did all SCID and nude recipients of normal thymus transplants. Morphological lesions similar to those found in scurfy mice occurred in all H-2-compatible nude and SCID recipients of sf/Y thymic grafts. Intraperitoneal injections of scurfy thymocytes, splenocytes, and lymph node cells also transmitted the scurfy phenotype to H-2-compatible nude mice and SCID mice. Our findings indicate that scurfy disease can be transmitted to T cell-deficient mice by engraftment of scurfy T cells, but that pathogenic scurfy T cell activities can be inhibited (or prevented) in immunocompetent recipient mice.

Feline lymphoid tissues engrafted into scid mice maintain morphologic structure and produce feline immunoglobulin.

To adapt the feline immune system to a small laboratory animal host, severe combined immunodeficient (scid) mice were engrafted with neonatal feline lymphoid tissues, including lymph node, thymus, spleen, and bone marrow. Lymph node and thymus tissue were implanted subcutaneously within the mammary fat pad, and a single-cell suspension of spleen, thymus, and bone marrow was inoculated intraperitoneally (IP). Seven groups of mice (three mice per group) were engrafted on day 0, and members of one group were euthanatized weekly from 2 to 8 weeks after engraftment. For each mouse, graft morphology was evaluated by light microscopy, feline DNA was detected in peripheral blood by polymerase chain reaction (PCR) amplification of feline-specific DNA sequences, and serum IgG concentration was measured by ELISA. Ten of 13 feline grafts evaluated histologically between 3 and 8 weeks after engraftment contained large focal aggregates of lymphocytes bordered by plasma cells. Of 14 thymus grafts evaluated histologically during the same period, 5 were characterized by dense accumulations of small lymphocytes surrounding thymic epithelial cells. Two of these thymus grafts were indistinguishable from age-matched feline thymus. At 2 weeks after engraftment, feline lymph node and thymus contained extensive central necrosis bordered by a narrow zone of lymphocytes and small-caliber blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Human hematolymphoid cells in SCID mice.

The severe combined immunodeficient C.B.-17 scid/scid (SCID) mouse has been widely used to study the normal processes of murine lymphoid differentiation. To create an in vivo model of the human hematolymphoid system, this mouse strain has been engrafted with human organ systems (the SCID-hu mouse) or with human peripheral blood mononuclear cells (the hu-PBL-SCID mouse). These mouse models have now been characterized and used to analyze human infectious diseases, hematopoiesis, malignancies and vaccines.