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.

Intrathecal application of neuroectodermally converted stem cells into a mouse model of ALS: limited intraparenchymal migration and survival narrows therapeutic effects.

Stem and progenitor cells provide a promising therapeutic strategy for amyotrophic lateral sclerosis (ALS). To comparatively evaluate the therapeutic potentials of human bone marrow-derived mesodermal stromal cells (hMSCs) and umbilical cord blood cells (hUBCs) in ALS, we transplanted hMSCs and hUBCs and their neuroectodermal derivatives (hMSC-NSCs and hUBC-NSCs) into the ALS mouse model over-expressing the G93A mutant of the human SOD1 gene. We used a standardized protocol similar to clinical studies by performing a power calculation to estimate sample size prior to transplantation, matching the treatment groups for gender and hSOD-G93A gene content, and applying a novel method for directly injecting 100,000 cells into the CSF (the cisterna magna). Ten days after transplantation we found many cells within the subarachnoidal space ranging from frontal basal cisterns back to the cisterna magna, but only a few cells around the spinal cord. hMSCs and hMSC-NSCs were also located within the Purkinje cell layer. Intrathecal cell application did not affect survival times of mice compared to controls. Consistently, time of disease onset and first pareses, death weight, and motor neuron count in lumbar spinal cord did not vary between treatment groups. Interestingly, transplantation of hMSCs led to an increase of pre-symptomatic motor performance compared to controls in female animals. The negative outcome of the present study is most likely due to insufficient cell numbers within the affected brain regions (mainly the spinal cord). Further experiments defining the optimal cell dose, time point and route of application and particularly strategies to improve the homing of transplanted cells towards the CNS region of interest are warranted to define the therapeutic potential of mesodermal stem cells for the treatment of ALS.

Neurogenic potential of human umbilical cord blood: neural-like stem cells depend on previous long-term culture conditions.

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB-NPs). Due to further expansion of these cells we have established the first human umbilical cord blood-derived neural-like stem cell line (HUCB-NSC) growing in serum-free (SF) or low-serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB-NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB-NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co-cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co-cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial-specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB-NSC on the neurogenic signals provided by surrounding environment in vitro.

Donor DNA can be detected in recipient tissues during rejection of allograft.

The main source of donor DNA in recipients of allograft are "passenger" cells. It is claimed that they are responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have observed that besides cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of the allograft. In this study, we provide evidence for the presence in the recipient of both DNA in "passenger cells" and free DNA in tissues at the terminal stage of rejection. Male BN (RT1 n) rat heart or skin was transplanted to female LEW (RT1 l) rats followed by a vascularized bone marrow in a hindlimb transplant. In another group, heart and skin were transplanted followed by immediate i.v. infusion of donor-type bone marrow cells. CsA was given in a dose of 17 mg/kg body weight for 30 days, then the rats were followed up until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and day 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, as well as lymphoid organs, at the time of rejection of allograft, but not when the rats were maintained on CsA. Taken together, donor DNAwas detected in recipient tissues at the time of heart or skin rejection. It appeared to be released from cells of rejecting grafts and not from "passenger" cells, representing only a minor cellular mass compared with the graft.

Vascularized bone marrow transplanted in orthotopic hind-limb stimulates hematopoietic recovery in total-body-irradiated rats.

Hematopoietic recovery after bone marrow transplantation (BMT) is reported to be slow with long-lasting immune deficiency. This may be attributable to lack of a proper microenvironment for hematopoietic cell proliferation and differentiation. We have designed a model in which complete hematopoietic reconstitution of lethally irradiated rats can be achieved by vascularized bone marrow transplantation (VBMT) in an orthotopic hind-limb graft. The aim of the study was to investigate the process of repopulation of bone marrow cavities and peripheral blood of irradiated rats after VBMT and, in particular, to follow the contribution of grafted BM cells and residual recipient BM cells in hematopoietic regeneration. Lewis hind-limbs were transplanted orthotopically to totally irradiated (8 Gy) syngeneic sex-mismatched recipients (VBMT). In the control group 8 x 10(7) BM cells in suspension were injected intravenously (BMCT). After 10 days BM and peripheral blood (PB) cells were collected from the recipient. For cell subset analysis cytomorphological evaluation of BM smears and flow cytometry of PB cells were performed. Additionally, PCR was performed using specific primers for rat Y chromosome (sex-determining region Y-Sry) to detect male (donor or recipient) cells in sex-mismatched BM graft recipients and the products were analyzed by electrophoresis. VBMT brought about much faster replenishment of nucleated cells in BM and PB than did BMCT. Cytometry analysis of PB cells revealed more lymphocytes in VBMT than in BMCT recipients. The amount of donor DNA of bands corresponding to Y-Sry was also higher in PB cells of VBMT than of BMCT recipients. The presence of host DNA was observed in PB cells of VBMT rats but was not detected in PB population of BMCT recipients. VBMT is highly effective in hematopoietic reconstitution of irradiated recipients. The fast cell maturation and repopulation may be due to the presence of stromal cells transplanted in a normal spatial relationship with donor hematopoietic cells in hind-limb graft. Self renewal of radioresistant host cells was seen after VBMT but not after BMCT.

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.

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.

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.

DNA from rejecting allografts can be detected in recipient nonlymphoid tissues.

The main source of donor DNA in recipients of allograft are "passenger" cells. They are claimed to be responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have noticed that beside of the cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of allograft. In this study we provide evidence for presence in the recipient of both, DNA in "passenger cells" and free DNA in tissues at terminal stage of rejection. Male BN (RTIn) rat heart or skin were transplanted to female LEW (RTII) rats followed by a vascularized bone marrow in hind-limb transplant. CsA was given in a dose of 17mg/kg b.w. for 30 days, then rats were followed until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, beside of lymphoid organs, at the time of rejection of allograft but not when rats were maintained on CsA. Taken together, donor DNA can be detected in recipient tissues at the time of heart or skin rejection. It seems to be released from cells of rejecting grafts and not from "passenger" cells representing only a minor cellular mass compared with the graft.

[Bacterial infections of skin and soft tissues in filariasis]. / Bakteryjne zakazenia skóry i tkanek miekkich w filariozie.

Adenolymphangitis is a common occurrence in filarial lymphedema. Damage to the lymphatics and lymph nodes by F. bancrofti is followed by obliteration of lymph vessels and lymph stasis. Obstruction of lymphatics prevents the bacteria penetrating skin to be evacuated with lymph stream to regional lymph nodes. Colonization of dermis, subcutis and lymphatics evokes clinical symptoms of adenolymphangitis. The question arises which strains of bacteria are responsible for the acute and chronic types of adenolymphangitis. The most probable strains responsible for this condition belong to the cocci and probably the bacillus strains.

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.

Cyclosporin A decreases lymphocyte migration to the heart allograft through suppression of their L-selectin expression.

Cyclosporin A (CsA) changes the distribution of the circulating pool of lymphocytes and decreases their traffic to organ allograft. The mechanism of this process is complex and includes, among others, inhibition of induction of nuclear factor of activated T cells and suppression of GM CSF and E-selectin expression. We studied the expression adhesion molecules CD11a, CD18, CD44, CD54 and CD62L on the thoracic duct lymphocytes of rats treated with CsA. The 7-day administration of CsA evidently decreased the expression of CD62L but did not affect the other adhesion molecules. Lower concentration of CD62L molecules on the surface of circulating lymphocytes may influence their migration to allograft and distribution in host lymphoid tissues.

Tolerance induction following allogeneic vascularized bone marrow transplantation--the possible role of microchimerism.

We have noticed that bone marrow transplanted in a vascularized limb graft, providing a continuous supply of donor bone marrow cells (BMC), may prolong the survival time of a skin graft from the same donor. The question arises whether the microchimerism raised plays a role in the prolonged survival of skin allografts. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection process of transplanted skin. Brown Norway (BN) rats served as donors and Lewis rats as recipients of VBMTx and free skin flap allografts. A 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 the monoclonal antibody OX27 directed against MHC class I polymorphic RT1 on BN cells and quantitatively analyzed in a FACStar. In the VBMTx group, the free skin flap survived 70 days after weaning off cyclosporine A (CsA). An intravenous infusion of BMC in suspension equivalent to that grafted in the 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 weaning off CsA but not in recipients of i.v. suspension BMC grafting.

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.

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.