SOD1/Rag2 Mice with Low Copy Number of SOD1 Gene as a New Long-Living Immunodeficient Model of ALS.

The most recent research concerning amyotrophic lateral sclerosis (ALS) emphasizes the role of glia in disease development. Thus, one can suspect that the effective therapeutic strategy in treatment of ALS would be replacement of defective glia. One of the basic problems with human glial progenitors (hGRPs) replacement strategies is the time needed for the cells to become fully functional in vivo. The lifespan of most popular high copy number SOD1 mutant mice might be too short to acknowledge benefits of transplanted cells. We focused on developing immunodeficient rag2-/- model of ALS with lower number of transgene copies and longer lifespan. The obtained hSOD1/rag2 double mutant mice have been characterized. QPCR analysis revealed that copy number of hSOD1 transgene varied in our colony (4-8 copies). The difference in transgene copy number may be translated to significant impact on the lifespan. The death of long- and short-living hSOD1/rag2 mice is preceded by muscular weakness as early as one month before death. Importantly, based on magnetic resonance imaging we identified that mutant mice demonstrated abnormalities within the medullar motor nuclei. To conclude, we developed long-living double mutant hSOD1/rag2 mice, which could be a promising model for testing therapeutic utility of human stem cells.

Fast lymphoid reconstitution after vascularized bone marrow transplantation in lethally irradiated rats.

BACKGROUND: Bone marrow (BM) transplantation for treatment of hematological and solid malignancies is routinely carried out in conjunction with radio- and chemotherapy. Many patients achieve complete remission of the malignant process; however, their lymphohematopoietic recovery remains in most cases incomplete. This is probably due to the functional changes in the recipient BM stromal cells subsequent to myeloablative therapy. Transplantation of BM hematopoietic cells in a spatial relationship with stromal cells would give an insight into the kinetics of hematological repopulation of the recipient. The aim of this study was to investigate the lymphopoietic reconstitution of irradiated rats after vascularized bone marrow transplantation (VBMT) in comparison with i.v. bone marrow cell (BMC) infusion. METHODS: Lewis rats were totally irradiated with 8Gy and repopulated with syngeneic BMC introduced i.v. or in orthotopic hind limb graft. Ten days after irradiation and BMC graft BM, peripheral blood (PB) and mesenteric lymph nodes (MLN) were collected. The yield and the phenotype of cells were analyzed. RESULTS: VBMT brings much higher cell repopulation of BM cavities of lethally irradiated rats than BMC infusion. Orthotopic hind limb graft promotes also rapid lymphocyte replenishment of PB and MLN of lethally irradiated syngeneic recipients. The population rate of BMC, PB lymphocytes, and MLN lymphocytes was higher after VBMT than BMC injection in suspension. The percentage of T and B lymphocytes in PB and MLN on day 10 after VBMT was comparable with control values. Reconstituted PB lymphocytes showed two subsets of CD4+ cells: "bright" and "dull." All CD4+ cells in PB lymphocytes of i.v. BMC infused recipients expressed low level of these molecules ("dull" subset). CONCLUSIONS: The results of our studies indicate that the presence of stromal cells in their close relationship with stem cells is essential for the fast lyphohematopoietic repopulation of irradiated recipients. The population of CD4+dull cells may represent immature cells. These cells were not found in MLN of VBMT rats. All MLN CD4+ cells represented the "bright" subset, what suggests that the process of cell maturation may occur in the lymphoid organs.

Rat liver contains a distinct blood-borne population of NK cells resistant to anti-asialo-GM1 antiserum.

A high level of anti-tumoral (NK) cytotoxicity was observed in the mononuclear population washed out from the liver microvasculature in rat. The level of cytotoxicity against K562 and YAC1 cells, percentage of large granular lymphocytes (LGL) and of OX8+ cells was significantly higher than in the inflowing portal and also caval blood. A low concentration of adherent, phagocytic and OX6+(Ia) cells and a higher level of cytotoxicity after removal of adherent cells ruled out the possibility of liver macrophages to be responsible for the cytotoxicity. Surprisingly, the liver-sequestered NK population turned to be resistant to anti-asialo-GM1 antiserum. Thus, liver microvasculature contains a significant number of cells with characteristics of NK cells which constitute a functionally distinct NK population.

Bacteriologic studies of skin, tissue fluid, lymph, and lymph nodes in patients with filarial lymphedema.

Filarial lymphedema is complicated by frequent episodes of dermatolymphangioadenitis (DLA). It is not certain whether DLA is of filarial or bacterial etiology. The frequency of episodic DLA does not depend on the presence or absence of microfilariae. Antibiotic therapy is effective in prevention and treatment of DLA. These observations point to the bacterial rather than filarial etiology of DLA. Skin and lymph node biopsies, tissue fluid, lymph, and blood from patients with chronic filarial lymphedema, and during acute episodes of DLA, were cultured for detection of bacteria. A high prevalence of bacterial isolates from the tissue fluid (64%), lymph (75%), and inguinal lymph nodes (66%) of limbs with filarial lymphedema was found. Bacillus cereus, Staphylococcus epidermidis, S. hominis, S. capitis, S. xylosus, and Micrococcus spp. were the most common isolates. Bacteria were also isolated from the blood of patients with recent episodes of DLA, with strains of the same phenotype and antibiotic sensitivity in all specimens from patients with DLA. Bacterial strains of the same phenotype and antibiotic sensitivity were documented on the toe web surface and in tissue fluid (25%), lymph (26%), or lymph nodes (41%). Increasing prevalence of bacterial isolates in tissue fluid, lymph, and lymph nodes was observed in advanced stages of lymphedema. Bacilli and cocci were sensitive to gentamicin, tetracyline, rifampicin, vancomycin, kanamycin and cotrimoxazole, and least sensitive to penicillin. Blood cultures of patients in the periods between DLA attacks were negative. In healthy controls without edema and episodes of DLA, tissue fluid did not contain bacteria. In lymph, only single colonies of Micrococcus and Acinetobacter were cultured in 12% of the cases. Impaired lymph drainage and lack of elimination of penetrating bacteria may be responsible for progression of lymphedema and recurrent attacks of DLA.

Bacteriological studies of blood, tissue fluid, lymph and lymph nodes in patients with acute dermatolymphangioadenitis (DLA) in course of 'filarial' lymphedema.

Filarial lymphedema is complicated by frequent episodes of dermatolymphangioadenitis (DLA). Severe systemic symptoms during attacks of DLA resemble those of septicemia. The question we asked was whether bacterial isolates can be found in the peripheral blood of patients during the episodes of DLA. Out of 100 patients referred to us with 'filarial' lymphedema 14 displayed acute and five subacute symptoms of DLA. All were on admission blood microfilariae negative but had a positive test in the past. Blood bacterial isolates were found in nine cases, four acute (21%) and five subacute (26%). In 10 acute cases blood cultures were found negative. Six blood isolates belonged to Bacilli, four to Cocci and one was Sarcina. To identify the sites of origin of bacterial dissemination, swabs taken from the calf skin biopsy wounds and tissue fluid, lymph and lymph node specimens were cultured. Swabs from the calf skin biopsy wound contained isolates in nine (47%) cases. They were Bacilli in nine, Cocci in three, Acinetobacter and Erwinia in two cases. Tissue fluid was collected from 10 patients and contained Bacilli in four (40%) and Staphylococci in three (30%). Lymph was drained in four patients and contained isolates in all samples (100%). They were Staphylococcus epidermis, xylosus and aureus, Acinetobacter, Bacillus subtilis and Sarcina. Three lymph nodes were biopsied and contained Staphylococcus chromogenes, xylosus, Enterococcus and Bacillus cereus. In six cases the same phenotypically defined species of bacteria were found in blood and limb tissues or fluids. In the 'control' group of patients with lymphedema without acute or subacute changes all blood cultures were negative. Interestingly, swabs from biopsy wound of these patients contained isolates in 80%, tissue fluid in 68%, lymph in 70% and lymph nodes in 58% of cases. In healthy controls, tissue fluid did not contain bacteria, and lymph isolates were found only in 12% of cases. This study demonstrates that patients with acute episodes of DLA reveal bacteremia in a high percentage of cases. Diversity of blood and tissue bacterial isolates in these patients points to a breakdown of the skin immune barrier in lymphedema and subsequently indiscriminate bacterial colonization of deep tissues and spread to an blood circulation.

Isolation and identification of rat Kupffer cells.

Preservation of liver for transplantation into allogeneic recipient affects the integrity and function of Kupffer cells. Rapid method for obtaining pure macrophages by dispersing the liver with collagenase and pronase would open possibility for different investigations. The experiments concerning morphology and phagocytic function were performed on Kupffer cells after 21 hours in vitro culture in Eagle medium with 20% inactivated calf serum. Studies on endocytosis using human red cells and colloidal carbon indicated that Kupffer cells revealed high phagocytic activity after culture.

C1 inactivator and C3b inactivator in peripheral lymph of normal men.

The concentrations of C1 inactivator and C3b inactivator were measured in the peripheral lymph of leg of normal men and compared with the concentrations in serum. Both inactivators were found in lymph, however, in concentrations lower than in serum (lymph/serum ratio for C1INA 0.236, for C3bINA 0.238). The levels of C1INA proteins in lymph were higher than of C1q and C1s (p less than 0.05), and of C3bINA higher or equal to those of C3 and C3PA. This may indicate, that the relatively high concentrations of both inactivators in the extravascular space, as compared with complement component proteins, can play a role in the control of activation process of complement in the interstitium and also explain to some extent the observed low hemolytic activity of lymph C1 and C3.

Storage of macrophages at -196 degrees C.

Oil induced mice peritoneal cells were stored at -196 degrees C for 3 months, using dimethyl sulfoxide (DMSO) as a cryoprotective medium. Recovery counts upon thawing were about 90%. Macrophages retained their phagocytic activity of SRBC in 89% and there were no significant differences in spontaneous migration, expression of membrane immune receptors and non-specific cytotoxicity of frozen-thawed cells compared with the controls.

Immunophenotypic and functional characterization of peripheral blood mononuclear cell populations in patients with dilated cardiomyopathy.

Cell-mediated immune responsiveness was evaluated in 8 patients with dilated cardiomyopathy (CDM). The percentage of blood OKM1 (monocytes), OKT3 (T-cells), OKT4 (helper cells), OKT8 (suppressor cells) and Leu7 (NK) positive cells remained within the normal range whereas the percentage of Leu11 (NK) and OKIa1 (Ia determinant) positive cells was found to be decreased. The natural killer (NK) cell cytotoxicity was significantly lowered. The ConA-suppressor cell activity was seen to be enhanced while the responsiveness of lymphocytes to ConA was diminished. Thus, no consistent numerical changes in lymphocyte subsets were observed in patients with CDM. Some functional deficiencies were, however, detected, and it is possible that these may play a role in the pathogenesis of CDM.

Inhibition of mouse bone marrow granulocyte-macrophage colony formation by factors derived from natural killer cells: an in vitro model for hybrid resistance.

Investigations were performed to study whether soluble factors produced by NK-cells could mediate "hybrid resistance" in vitro. NK-cells enriched from spleens of B6D2F1 hybrid mice were incubated with parental B6 bone marrow, and the effect of the derived supernatants on the development of granulocyte-macrophage colony forming cells (GM-CFC) was assessed. Cell free supernatants obtained from low density cells (LDC) of B6D2F1 hybrids stimulated with bone marrow cells (BMC) from B6 mice inhibited GM-CFC formation. The inhibition was similar using B6, D2 or B6D2F1 bone marrow cells as the targets for GM-CFC growth. Our findings suggest that NK cells from F1 hybrid mice when stimulated with BMC from B6 mice release inhibitory factors, different from IFN-gamma and that this production may represent a mechanism of natural resistance to parental H-2b bone marrow grafts.

Liver sinusoidal washout cells transferred to allogeneic recipients reveal tolerogenic properties.

Liver sinusoidal washout cells, containing a large fraction of LGL cytotoxic to tumor cell lines in vitro, are less responsive to mitogens and suppress the responsiveness of autologous and third-party PBM to mitogens and autologous mixed lymphocyte culture, compared with autologous portal vein or PBM. They are less immunogenic after IV administration into allogeneic recipients, which can be observed in an in vivo allogeneic lymphocyte cytotoxicity test. Immunization of allogeneic recipients with liver sinusoidal washout cells slightly prolongs heart allograft survival time.

Liver sinusoidal passenger lymphocytes reveal suppressive properties and prolong allograft survival.

Liver sinusoidal washout cells are low responders to mitogens, and suppressive in autologous PBM, and portal blood mononuclear cells, PHA supplemented, and autologous mixed lymphocyte cultures. They are less immunogenic after IV administration into allogeneic recipients than PBM, which could be measured in an in vivo allogeneic lymphocyte elimination test. The level of immunization is strain dependent. In the BN to LEW combination, they are more immunogenic than in the LEW to DA pairs. This parallels heart allograft survival times, which were longer in LEW to DA than BN to LEW combinations.

Natural killer lymphocytes in rat liver sinusoidal blood.

Blood-borne cells with the characteristics described for the natural killer (NK) lymphocytes were recovered from the liver sinusoids in rat. They had cytotoxic activity against K 562 cells in a 18 h 51Cr release assay, target binding rate and large granular lymphocytes percentage higher than the inflowing portal blood cells. The cytotoxic effector population was non-adherent and peroxidase negative, enriched in cells with Fc- and C36-receptors. Deprivation of liver of portal or mesenteric blood supply significantly decreased the liver NK activity not affecting the level of cytotoxicity in the portal blood. Splenectomy produced only a moderate decrease of activity. These findings may indicate that liver sinusoids is the site of accumulation of blood-born NK cells or of maturation of the NK-precursors. One possible mechanism by which natural cytotoxicity controls tumor growth and spread might be the killing of circulating tumor cells arrested in the liver by the blood-born NK-cells.

Immunologic characteristics of human peripheral lymph cell populations.

Cell populations in afferent lymph of human leg were defined by surface characteristics and cytotoxic activity in 7 normal men and 9 patients with localized cancer. A higher percentage of E-rosette forming cells was found in lymph (78.5 and 83.0) than in blood (60.0 and 63.0 p less than 0.05). The percentages of lymph EA-RFC were 10.3 and 18.0, of EAC-RFC 13.1 and 8.0, of surface immunoglobulin carrying cells 3.0 and 3.1. In blood 20.6 and 18.0 percent of cells formed EA-rosettes, 23.0 and 15.6 EAC-rosettes, 5 and 9.5 contained surface immunoglobulins. The differences between lymph and blood EA- and EAC-RFC in normals were statistically significant (p less than 0.05). In cancer patients only lymph-blood differences for S Ig+ were significant (p less than 0.05). No significant differences were found between normals and cancer patients. In both groups, the natural cytotoxicity against K 562 cells was 6 times lower in lymph as compared to blood (p less than 0.05), the cytotoxicity in those with cancer was higher than in normals (p less than 0.05). The study indicates that B cells have a limited tendency toward leaving the blood circulation and migrating through tissues. Moreover natural killer cells do not seem to belong to the recirculating pool of lymphocytes.

Immune proteins in peripheral tissue fluid-lymph in patients with filarial lymphedema of the lower limbs.

Immune proteins and cytokine concentrations and activity were measured in skin tissue fluid-lymph and blood serum of patients with lower leg filarial lymphedema. High levels of lymph gamma-globulins, alpha-1-acid glycoprotein and IgG lymph/serum ratio were found. Lymph from filarial patients had an elevated lymph stimulatory effect on blood mononuclear cell culture with phytohemagglutinin. There was also a high concentration and activity of IL-1 beta but gamma-interferon was not detected. The deranged pattern of immune proteins and high activity of IL-1 suggest persistence of an ongoing local inflammatory process despite the absence of overt dermatitis. The high tissue-lymph IL-1 concentration and activity may signify a cytokine network promoting keratinocyte and fibroblast proliferation commonly seen on skin histology in patients with filariasis.

Reconstitution of lymphoid tissue after vascularized bone marrow transplantation.

We reported previously that vascularized bone marrow transplantation (VBMT) in an orthotopic hind limb graft brings about complete repopulation of bone marrow cavities in lethally irradiated syngeneic recipients within 10 days. Intravenous infusion of an equivalent volume of bone marrow cell suspension was evidently less effective. The purpose of this study was to investigate the reconstitution of immunocompetent compartments of lethally irradiated syngeneic rats after VBMT. Lewis rat hind limbs were transplanted orthotopically into irradiated recipients. Ten days after irradiation and bone marrow transplantation, bone marrow, mesenteric lymph nodes and sera from rats were harvested. Responsiveness of mesenteric lymph node lymphocytes (MLNL) to mitogens and lymphocyte proliferation in the presence of sera and bone marrow cell (BMC) culture supernatants was measured. Our studies have shown that vascularized bone marrow transplantation brings about rapid replenishment of lymphoid organs of lethally irradiated syngeneic recipients. The repopulating subsets were fully responsive to mitogens. Sera from reconstituting rats had no evident effect on proliferation of mature lymphocytes. Intravenous infusion of BMC in suspension, in a number equivalent to that grafted in hind limb transplant, was less efficient in reconstituting lymphoid tissue.

Sinusoidal lymphocytes of liver graft may limit the recurrence of hepatic tumor in liver transplant recipients.

The liver immune function is associated with specific lymphocyte population transiently marginated in the liver sinusoids. These cells are of blood origin, however they are phenotypically and functionally different from peripheral blood lymphocytes. The question arises whether tumor proliferating in the liver can modify cell recruitment and function of marginating sinusoidal lymphocytes. Studies were performed in Wistar rats. Livers of normal and colon cancer (induced by i.v. injection of CC531 cells) metastases bearing rats were perfused for sinusoidal lymphocyte isolation. Our studies showed no difference between the number of lymphocytes retained in sinusoids of tumor bearing and normal rats. T lymphocyte subsets remained in similar proportions in liver with colorectal metastases as in normal rats. Long lasting presence of tumor in the liver was accompanied by decreased cytotoxic activity of liver sinusoidal lymphocytes, whereas it had no influence on cytotoxicity of peripheral blood lymphocytes. Repopulation of tumor liver with peripheral blood cells restored cytotoxic activity of sinusoidal lymphocytes.

Kinetics of bone marrow repopulation in lethally irradiated rats after transplantation of vascularized bone marrow in syngeneic hind limb.

Grafting of the recipient with bone marrow cell suspension provides only few stromal cells and no autologous environment for cooperation between hemopoietic and stromal cells. Vascularized bone marrow grafts provide the recipient with bone marrow hemopoietic and stromal cells in their natural spatial relationship. It is expected that such a graft will resume its function soon after transplantation and almost immediately repopulate bone marrow cavities of the irradiated recipient as well as supply the sites of cytopoiesis with the necessary growth factors. The aim of the study was to investigate the process of repopulation of bone marrow cavities and lymphoid organs of irradiated recipient by bone marrow cells from syngeneic hind limb transplant. Lewis rats received total body irradiation of 8 Gy followed by orthotopic transplantation of syngeneic limb or i.v. infusion of equivalent amount of bone marrow cells. In control experiments the hind limb was shielded with lead plate during total body irradiation. Ten days after irradiation and hind limb transplantation the yield of nucleated cells from tibia was reaching values of normal animals. In rats receiving i.v. bone marrow cell infusion it was 40% of control values and in rats repopulated from shielded own hind limb it was 60% of controls. In all groups a higher percentage of early and immediate normoblasts and a reduced pool of juvenile and segmented neutrophils was observed. Thirty days after irradiation and repopulation procedures all parameters were returning to normal levels in each group. The results indicate that bone marrow cell transplantation in hind limb graft is highly effective in lethally irradiated animals in reconstituting bone marrow.

Body distribution of syngeneic and allogeneic cells from vascularized bone marrow graft.

In the previous studies we showed that vascularized bone marrow graft (VBMtx) in transplanted rat hind limb brings about complete repopulation of syngeneic recipient BM cavities and lymphoid organs within 10 days. Transplantation of an equivalent number of bone marrow cells (BMC) in suspension did not produce repopulation until day 30. In this study we present data on transplantation of allogeneic VBM and compare them with those obtained in a syngeneic combination. In the LEW or BN to LEW combination BM cells were labelled with 51Cr, injected i.v., 24 h later the hind limb was amputated and transplanted to a LEW rat. BM cells emigrated from the transplanted limb to the recipient and distributed in BM cavities and lymphoid tissues. In the LEW to LEW combination the level of radioactivity in recipient tibia was after 24 h 0.85, in spleen 2.43, in mesenteric lymph node 0.52%/g of tissue, whereas in the BN to LEW model it was 0.11, 1.83 and 0.15%, respectively. The calculated numbers of BM cells which populated recipient tissues were 8-10-times lower in the allogeneic compared with syngeneic combination. This was probably due to the nonspecific elimination of some subsets of BM cells (allogeneic BMC cytotoxicity). Administration of anti-asialo GMI antiserum to the recipient abrogated the cytotoxic effect. Taken together, major differences in kinetics of seeding and repopulation of BMC from VBMTx were found. Elimination of recipient NK cells with AAGMI antiserum attenuated the nonspecific cytotoxic effect. This protocol allows protection of the grafted BMC and increases the efficacy of the transplant.

Microchimerism following allogeneic vascularized bone marrow transplantation--its possible role in induction of posttransplantation tolerance.

We have noticed that bone marrow transplanted in a vascularized limb graft providing a continuous supply of donor BMC may prolong the survival time of skin graft from the same donor. The question arises whether the raised microchimerism plays a role in the prolonged survival of skin allograft. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection processes of transplanted skin. The BN rats served as donors and LEW rats as recipients of VBMTx and free skin flap allograft. Hind limb was transplanted followed by a full-thickness skin graft on the dorsum. Cellular microchimerism was investigated in recipients of VBMTx and skin grafts in blood, spleen, mesenteric lymph node and bone marrow with monoclonal antibody OX27 directed against MHC class I polymorthic RTI on BN cells and quantitatively analysed in FACStar. In VBMTx group free skin flap survived 70 days after weaning of CsA. Intravenous infusion of BMC in suspension equivalent to that grafted in hind limb did not prolong skin graft survival after cessation of CsA therapy. Donor-derived cells could be detected in VBMTx recipients as long 70 days after wearing of CsA but not in recipients of i.v. suspension BMC grafting.