Vladimír Holáň

Nanofibers prepared by needleless electrospinning technology as scaffolds for wound healing

Electrospun gelatin and poly-ε-caprolactone (PCL) nanofibers were prepared using needleless technology and their biocompatibility and therapeutic efficacy have been characterized in vitro in cell cultures and in an experimental model of a skin wound. Human dermal fibroblasts, keratinocytes and mesenchymal stem cells seeded on the nanofibers revealed that both nanofibers promoted cell adhesion and proliferation. The effect of nanofibers on wound healing was examined using a full thickness wound model in rats and compared with a standard control treatment with gauze. Significantly faster wound closure was found with gelatin after 5 and 10xa0days of treatment, but no enhancement with PCL nanofibers was observed. Histological analysis revealed enhanced epithelialisation, increased depth of granulation tissue and increased density of myofibroblasts in the wound area with gelatin nanofibers. The results show that gelatin nanofibers produced by needleless technology accelerate wound healing and may be suitable as a scaffold for cell transfer and skin regeneration.

Separation of multiple genes controlling the T-cell proliferative response to IL-2 and anti-CD3 using recombinant congenic strains.

T lymphocytes of the strain BALB/cHeA exhibit a low proliferative response to IL-2 and a high response to the anti-CD3 monoclonal antibodies, while the strain STS/A lymphocyte response to these stimuli is the opposite. We analyzed the genetic basis of this strain difference, using a novel genetic tool: the recombinant congenic strains (RCS). Twenty BALB/c-c-STS/Dem (CcS/Dem) RCS were used, each containing a different random set of approximately 12.5% of the genes from STS and the remainder from BALB/c. Consequently, the genes participating in the multigenic control of a phenotypic difference between BALB/c and STS become separated into different CcS strains where they can be studied individually. The strain distribution patterns of the proliferative responses to IL-2 and anti-CD3 in the CcS strains are different, showing that different genes are involved. The large differences between individual CcS strains in response to IL-2 or anti-CD3 indicate that both reactions are controlled by a limited number of genes with a relatively large effect. The high proliferative response to IL-2 is a dominant characteristic. It is not caused by a larger major cell subset size, nor by a higher level of IL-2R expression. The response to anti-CD3 is known to be controlled by polymorphism in Fcγ receptor 2 (Fcgr2) and the CcS strains carrying the low responder Fcgr2 allele indeed responded weakly. However, as these strains do respond to immobilized anti-CD3, while the STS strain does not, and as some CcS strains with the BALB/c allele of Fcgr2 are also low responders, additional gene(s) of the STS strain strongly depress the anti-CD3 response. In a backcross between the high responder and the low responder strains CcS-9 and CcS-11, one of these unknown genes was mapped to the chromosome 10 near D10Mit14. The CcS mouse strains which carry the STS alleles of genes controlling the proliferative response to IL-2 and anti-CD3 allow the future mapping, cloning, and functional analysis of these genes and the study of their biological effects in vivo.

Augmented production of proinflammatory cytokines and accelerated allotransplantation reactions in heroin-treated mice

Heroin treatment or abusive drug addiction influences many physiological functions, including the reactions of the immune system. Although suppression of various manifestations of the immune system after heroin (or morphine) administration has been reported, we show here that production of proinflammatory cytokines and nitric oxide (NO) was enhanced and allotransplantation reactions were accelerated significantly in heroin‐treated recipients. Mice were treated by a subcutaneous administration of heroin (diacetylmorphine) given in one or repeated daily doses. The ability of spleen cells from treated mice to respond in vitro to alloantigens and to produce IL‐2, IL‐4, IL‐10 and IFN‐γ, and the production of IL‐1β, IL‐12 and NO by peritoneal macrophages, were tested. Within 2 h after heroin administration, proliferative responses to alloantigens and the production of IL‐1β, IFN‐γ, IL‐12 and NO were enhanced significantly. In contrast, the production of anti‐inflammatory cytokines IL‐4 and IL‐10 was at the same time rather decreased. As a consequence, skin allografts in heroin‐treated mice were rejected more promptly than in untreated or vehicle‐treated recipients. Similarly, the growth of allogeneic tumours induced by high doses of tumour cells was suppressed significantly in heroin‐treated mice. The enhancing effects of heroin on the production of proinflammatory cytokines were antagonized by naltrexone, a specific inhibitor of classic opioid receptors. These results show that heroin treatment augments production of proinflammatory cytokines and accelerates allotransplantation reactions. The observations thus illustrate the complexity of the effects of heroin on the immune system and should be taken into account during medical treatment of opiate addicts and in the use of morphine to decrease pain in various clinical situations.

Characterization of human adipose tissue-derived stromal cells isolated from diabetic patient's distal limbs with critical ischemia

Adipose tissue is an abundant source of autologous adult stem cells that may bring new therapeutic perspectives on the treatment of diabetes and its complications. It is unclear whether adipose tissue‐derived stromal cells (ASCs) of diabetic patients, constantly influenced by hyperglycaemia, have the same properties as non‐diabetic controls. As an alternative source of ASCs, adipose tissue from distal limbs of diabetic patients with critical ischemia was isolated. ASCs were characterized in terms of cell surface markers, multilineage differentiation and the expression of vascular endothelial growth factor (VEGFA), chemokine‐related genes and compared with non‐diabetic controls. Flow cytometry analysis confirmed mesenchymal phenotypes in both diabetic and non‐diabetic ASCs. Nevertheless, 40% of diabetic and 20% of non‐diabetic ASC samples displayed high expressions of fibroblast marker, which inversely correlated with the expression of CD105. In diabetic patients, significantly decreased expression of VEGFA and chemokine receptor CXCR4 was found in fibroblast‐positive ASCs, compared with their fibroblast‐negative counterparts. Reduced osteogenic differentiation and the downregulation of chemokine CXCL12 were found in fibroblast‐negative diabetic ASCs. Both diabetic and non‐diabetic ASCs were differentiated into adipocytes and chondrocytes and did not reveal islet‐like cell differentiation. According to this study, adipose tissue from distal limbs of diabetic patients is not satisfactory as an autologous ASC source. Copyright

Identical genetic control of MLC reactivity to different MHC incompatibilities, independent of production of and response to IL-2

The inbred strain STS/A exhibits a higher proliferative response in the mixed lymphocyte culture (MLC) to stimulator cells of all 11 tested inbred mouse strains with 10 different major histocompatibility complex (MHC) haplotypes, as well as to stimulation with IL-2 than does the strain BALB/cHeA. However, alloantigen-stimulated BALB/c cells produce more IL-2 than STS/A cells. To study the genetic basis of these differences, we used 20 recombinant congenic strains (RCS) of the CcS/Dem series. Each of these CcS/Dem RC strains contains a different subset of about 12.5% of genes from the STS/A strain and the remaining approximately 87.5% of BALB/c origin genes. As a result the multiple non-linked genes responsible for phenotypic differences between BALB/c and STS/A became separated into different CcS/Dem strains. The strain distribution pattern (SDP) of high or low MLC response of individual CcS/Dem strains to stimulator cells of four different strains was almost identical, indicating that differences in responsiveness, rather than the alloantigenic difference itself, determine the magnitude of the response, and that the responsiveness to different alloantigens is largely controlled by the same genes. The SDP of IL-2 stimulation was different from that of MLC responsiveness. The differences in the proliferative responses observed among individual CcS/Dem strains were not due to differences in numbers of CD3+, CD4+ or CD8+ cells or to the observed differences in IL-2 production, and hence they likely reflect genetically determined intrinsic properties of T cells. These results show that a set of non-linked genes controls proliferative responses in MLC irrespective of the MHC haplotype of the stimulator cells, and that stimulation with IL-2 and production of IL-2 are controlled by different subsets of genes. Since the genomes of all RCS are extensively characterized by microsatellite markers, they can be used to map the genes controlling proliferative responsiveness to stimulation with alloantigens and IL-2.

Effects of tumor necrosis factor α, interferon α and interferon γ on non-lymphoid leukemia cell lines: Growth inhibition, differentiation induction and drug sensitivity modulation

SummaryThe potential role of tumor necrosis factor α (TNFα), interferon α (IFNα) and interferon γ (IFNγ) in the therapy of non-lymphoid leukemia was studied in ten non-lymphoid leukemia cell lines. All three cytokines tested inhibited the growth of the cell lines. However, a high degree of variability in susceptibility to cytotoxic/cytostatic effect of the cytokines was found among individual cell lines. Some cell lines were sensitive to the antiproliferative action of only one of the cytokines tested, but were resistant to the others. Combinations of two cytokines had additive or synergistic effects and inhibited cell growth to a greater extent than did the individual cytokines alone. In addition to the growth-inhibitory effect, the cytokines induced an apparent cell differentiation. The differentiation of the two most sensitive cell lines, EoL-1 and PL-21, was confirmed using the nitroblue tetrazolium reduction test, by changes in cell morphology, immunophenotype marker profiles and by changes in c-myb expression. Furthermore, we showed that even in the cell lines relatively resistant to the antiproliferative effect of cytokines, such as cell line KCL-22, the inhibition of cell growth could be markedly increased with the DNA-topoisomerase-II-targeted drug, doxorubicin. Our data thus suggest that TNFα, IFNα and IFNγ together have a potential role in the immunotherapy of non-lymphoid leukemia in terms of their antiproliferative action, and their ability to induce differentiation and to modulate drug sensitivity.

Genetic control of T-cell proliferative response in mice linked to chromosomes 11 and 15

Antigen-induced activation of T lymphocytes plays a central role in specific immune reactions. This response is influenced by antigen presenting cells and depends on T-cell signalling pathways (Wegener et al. 1992; Jenkins and Johnson 1993; Howe and Weiss 1995). T cells can be stimulated to produce cytokines and cytokine receptors which drive proliferation and differentiation, and to express cell surface and intracellular molecules involved in effector functions (Miyajima et al. 1992). As intracellular events initiated by the ligation of theα/β heterodimer of the T-cell receptor (TCR) are mediated by signal transduction through the CD3 complex, the CD3 monoclonal antibody (mAb) is widely used as a trigger for the analysis of T-cell activation. Mouse strains BALB/cHeA (BALB/c) and STS/A (STS) differ in their response to CD3 antibody; lymphocytes of the strain BALB/c are high responders, whereas STS lymphocytes do not respond to this stimulus. This deficient response is due to the STS allele at the Fcgr2 locus which produces a non-functional CD32 (Fc γ receptor 2) molecule (Hibbs et al. 1985; Lipoldova ́ et al. 1995). To analyze whether other gene(s) influence the response to mAb CD3 as well, we used the recombinant congenic strains (RCS) of the BALB/c-c-STS/Dem (CcS/Dem) series, which have been developed for genetic analysis of multigenically-controlled complex biological processes (Demant and Hart 1986; Demant 1992). A series of RCS comprises 20 homozygous strains, all produced by backcrossing and inbreeding from two parental inbred strains, a background strain, and a donor strain. Each CcS/Dem strain of the series contains a different, random set of approximately 12.5% genes from the donor strain STS and approximately 87.5% genes from the background strain BALB/c. RCS have been succesfully used in studies of genetics of tumor susceptibility (Moen et al. 1991; Fijneman et al. 1994), apoptosis (Mori et al. 1995), and the immune response (Lipoldova ́ et al. 1995; Holáň et al. 1996). In our previous work (Lipoldová et al. 1995) we observed large differences in the ability of CcS/Dem strains to respond by proliferation to mAb CD3. The donor strain STS is a non-responder because of its non-functional Fcgr2 allele, as are the strains CcS-11 and -12 which carry this allele. However, additional genes are involved, because some RCS strains are even higher responders than the strain BALB/c, indicating that they received high-response alleles at some unknown loci from the non-responder strain STS, in which the effect of these high-response genes is not visible because of the nonfunctional allele atFcgr2 and the presence of low-response alleles at additional loci (Lipoldova ́ et al. 1995). In order to map some of these genes, we prepared F 2 hybrids between one of the high responder RC strains, CcS-4, and the parental strain BALB/c. Three-hundred-and-forty mice, 6 to 14 weeks old, both male and female, were analyzed. The mice were shipped from Amsterdam to Prague in two separate groups (sets) with a three-month interval. Each day in a separate experiment 20 F 2 mice and appropriate controls were tested. The mAb KT3 recognizing the CD3 ε chain was kindly provided by K. Tomonari (Tomonari et al. 1988). The proliferative response of spleen cells to soluble mAb CD3 was tested as described previously (Lipoldova ́ et al. 1995). Counts indicating 3H-incorporation in anti-CD3stimulated splenocytes were divided by the counts obtained when the same cells were maintained in medium only. These ratios were logarithmically transformed to obtain a normal distribution. The difference between the stimulated a d control cultures was also used and gave essentially the same results (data not shown). The strain CcS-4 carries genetic material of STS origin on eight chromosomes (Stassen et al. 1996). These segments were typed in the F2 hybrids by 23 microsatellite markers using the polymerase chain reaction (Dietrich et al. 1992). Evaluation of linkage was performed by analysis of variance (ANOVA, NCSS), using the marker, gender, and age as fixed factors and the day of experiment as a random factor. The results were confirmed by using a mixed model analysis of H. Havelková? M. Krulová ? M. Kosařová ? V. Holáň M. Lipoldová ( ) Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Fleming. na ́m. 2, 16637 Praha 6, Czech Republic

Immunosuppressive effects of vermiculine in vitro and in allotransplantation system in vivo.

Vermiculine, a macrocyclic aglycosidic dilactone isolated from Penicillium vermiculatum, has been shown to have immunomodulatory properties. Here, we tested the effects of vermiculine on selected parameters of cell-mediated immunity in vitro and on skin allograft survival in vivo. Vermiculine inhibited in a dose-dependent manner the proliferation of mouse spleen cells stimulated with Concanavalin A ((Con A), i.e. T-cell mitogen), bacterial lipopolysaccharide ((LPS), B-cell mitogen) or with irradiated allogeneic cells. In addition, vermiculine dose-dependently inhibited the production of Thl (IL-2, IFN-gamma) and Th2 (IL-4, IL-10) cytokines and suppressed the production of nitric oxide (NO) by activated macrophages. When compared with cyclosporine (CsA), vermiculine was less inhibitory for IL-2 gene expression and IL-2 synthesis, comparably suppressive on IL-10 production and even more inhibitory for NO synthesis. These observations suggest that vermiculine and CsA inhibit immune reactions by different mechanisms. Treatment of graft recipients with vermiculine or CsA prolonged survival of skin allografts in a mouse model. The combination of both drugs enhanced the survival of allografts significantly more than either drug alone. The results thus suggest that vermiculine is a potential immunosuppressive drug acting by a mechanism distinct from that of CsA, and thus it may be used alone or in combination with other drugs for immunoregulatory purposes.

T-cell proliferative response is controlled by locus Tria3 on mouse chromosome 17

Immunogenetics (1999) 49:235–237 Q Springer-Verlag 1999H. Havelkova 7 M. Kosarˇova 7 M. Krulova 7 V. HolanˇM. Lipoldova (Y)Institute of Molecular Genetics, Academy of Sciences of theCzech Republic, Fleming. nam. 2, 166 37 Praha 6,Czech Republic,e-mail: Lipoldova@img.cas.cz,Tel.: c4202-243-10-195, Fax: c4202-243-10-995P. DemantDivision of Molecular Genetics, The Netherlands CancerInstitute, Amsterdam, The Netherlands

Mouse model for analysis of non-MHC genes that influence allogeneic response: recombinant congenic strains of OcB/Dem series that carry identical H2 locus

Alloreactivity is the strongest known primary immune response. Its clinical manifestations are graft rejection, graft-versus-host disease and graft-versus-leukemia effect. The strongest stimulation by allogeneic cells is due to incompatibility at the major histocompatibility complex (MHC) genes. However, the non-MHC genes also participate in allogeneic response. Here we present a mouse model for study of the role of non-MHC genes in regulation of alloreactivity and show that they besides encoding antigens also regulate the responsiveness. Recombinant congenic strains (RCS) of O20/A (O20)-c-B10.O20/Dem (OcB/Dem) series have been derived from the parental strains O20 and B10.O20, which carry identical MHC haplotypes (H2pz) and therefore their differences in alloantigen response depend only on non-MHC genes. We have tested a MLR response by spleen cells of the strains O20, B10.O20, and 16 OcB/Dem strains through stimulation by cells from strains C57BL/10 (H2b), BALB/c (H2d), CBA (H2k), and DBA/1 (H2q) alloantigens. Proliferative response of O20, B10.O20 and OcB/Dem strains to these four alloantigens exhibited a similar but not completely identical pattern of reactivity. The responses to different alloantigens were highly correlated: C57BL/10-BALB/c r = 0.87, C57BL/10-CBA r = 0.84, C57BL/10-DBA/1 r = 0.83. Cluster analysis of the responses by O20, B10.O20, and OcB mice identified groups of strains with distinct patterns of response. This data shows that two main types of genes influence MLR: 1. structural genes for major and minor alloantigens and 2. genes regulating T-cell receptor signal transduction or mediating costimulatory signals by antigen-presenting cells.