Transplantation of keratinocyte spore-like stem cells.

BACKGROUND: Wound granulation tissue should be covered by epidermal cells migrating from the basal layer of the epidermis or hair "bulge" of the wound edge. However, new epidermal islands are frequently formed on the granulation tissue remote from the wound edge. Thus, current theory of "bulge"-originating stem cells does not necessarily correspond to the histological pictures of the healing wound. We took imprints of a leg ulcer surface and found single dispersed, large nucleated cells, some of them in mitosis. These cells resembled those from epidermal spinosum layer. The question arouse as to whether these cells might be the "spore-like" stem cells creating epidermal island. We found similarly shaped cells among the keratinocyte preserved in pulverized sodium chloride as the only surviving population in culture and revealing enzymatic activity. The aim of this work was to study whether the population of human keratinocytes surviving sodium chloride preservation and transplanted to SCID mice may form epidermis. METHODS: The 12-month sodium chloride-preserved and cultured keratinocytes (KC) were transplanted to the wound on the dorsum of SCID mice for 14 and 21 days. RESULTS: Ninety-five percent of cultured KC were enzymatically active "large" cells; they did not express p63 and CD29 claimed as specific for stem cells, and they did not proliferate. Transplanted to the center of the wound, they formed small KC islands and became confluent after 14 days. CONCLUSIONS: The "large" epidermal keratinocytes survived the 12-month preservation in anhydrous sodium chloride. Transplanted to the wound, they formed epidermal islands of human phenotype. These cells may be the so-called "spore-like" stem cells.

Skin own bacteria may aggravate inflammatory and occlusive changes in atherosclerotic arteries of lower limbs.

AIM: Seroepidemiological studies have given rise to the hypothesis that microorganisms like Chlamydia pneumoniae (CP), Helicobacter pylori (HP), cytomegalovirus (CMV), HCV types 1 and 2, and bacteria involved in dental or other unspecified infection sites may initiate or maintain the atherosclerotic process in lower limb arteries. However, not much attention has been attached to the patient's own limb skin and deep tissues bacterial flora, activated in ischemic tissues. This flora may enhance the inflammatory and thrombotic process in the atherosclerotic arteries. Lower limb tissues are exposed to microorganisms from the environment (foot) and microbes on floating epidermal cells from the perineal and anal regions. The aim of this paper was to identify microbial cells and their DNA in perivascular tissues and arterial walls of lower limbs. METHODS: Bacterial cultures and PCR method for detection of 16sRNA and immunohistopathological staining for identification of immune cells infiltrating vascular bundles. RESULTS: 1) specimens of atherosclerotic calf and femoral arteries contained bacterial isolates and/or their DNA, whereas, in control normal cadaveric organ donors' limb arteries or patients' carotid arteries and aorta bacteria they were detected only sporadically; 2) lower limb lymphatics contained bacterial cells in 76% of specimens, whereas controls only in 10%; 3) isolates from limb arteries and lymphatics belonged in majority to the coagulase-negative staphylococci and S.aureus, however, other highly pathogenic strains were also detected; 4) immunohistopathological evaluation arterial walls showed dense focal infiltrates of granulocytes and macrophages. CONCLUSION: Own bacterial isolates can be responsible for dense neutrophil and macrophage inflitrates of atherosclerotic walls and periarterial tissue in lower limbs and aggravate the ischemic changes.

Differentiation of cellular reaction to alloantigens and bacterial infection in human skin graft--immunosuppressive drugs or antibiotics.

The human hand transplantations prompted revival of interest in evaluation of the rejection process of the grafted skin and its control with the antirejection drugs [1-3]. In case of first hand transplantation a combined immunosuppressive regimen was applied with currently available drugs resulting in acceptance of the entire composite graft. No major untoward systemic effects of antirejection therapy were observed. The most important clinical conclusion was that allogeneic skin can be accepted and function as in a normal extremity, although the attack of host cells on the graft can not be totally eliminated. Chronic perivascular and subepidermal infiltrates with recipient cells could be seen [4]. Another problem connected with skin transplantation is graft infection. Skin is inhabited by a specific spectrum of bacteria [5]. Allografted skin is more sensitive to bacterial penetration than normal skin due to local damage by the host-versus-graft cellular reaction and compromised immune reactivity to bacterial antigens by the immunosuppressive therapy. The histological pictures of rejecting skin represent a mixture of cellular reaction against the graft and penetrating microbes. Alloreaction requires modification of immunosuppressive regimen and infection is an indication for prolonged antibiotic therapy against skin bacterial flora. The question arises how to discriminate the alloreactive and bacterial changes in the skin graft. We studied the histological pictures of rejecting and infected human skin after transplantion to scid mice.

Graft-induced polyclonal activation of B cells. I. Alloantigen-induced humoral response in mice.

In recent years evidence has accumulated pointing out that graft rejection may be mediated not only by cellular but also humoral mechanisms. To study this problem in some detail, humoral responses were studied in mice subjected to skin grafting across the H-2 locus. B cell reactivity was measured during the first and second set reactions on consecutive days after transplantation until graft rejection. In vitro B cell activity was assessed by means of the reverse plaque-forming cell assay (PFC) using cells obtained from the lymph nodes and spleens. The results indicate that polyclonal activation of B cells is an integral part of the immune response to skin transplantation in mice. However, no correlation was found between the level of activation as demonstrated by the PFC and the time of graft rejection.

Lymphatic system of mice born from mothers treated with ampicillin or cloxacillin during gestation.

Ampicillin, an antibiotic, widely used for combating bacterial infections exerts great influence on cells of the immune system of mouse and man. We have studied the effect of ampicillin and cloxacillin treatment of mice in the final week of pregnancy on the development of the lymphatic system of their offsprings. The mice born from antibiotic or saline treated mothers were examined 30 days after delivery. The examination of relative organ weight, cellularity and histopathological picture of lymphatic system (thymus, spleen and lymph nodes) and some other organs was performed. In the group of mice from ampicillin treated mothers we have found decreased relative weight of thymus and spleen and increased weight of lymph nodes with increased cellularity in thymus and lymph nodes. In the group of mice from cloxacillin treated mothers increased cellularity of thymus and lymph nodes was found. The histopathological study of lymphatic organs structure did not reveal any specific changes but the symptoms of focal degeneration and fat necrosis were found in livers of mice born from ampicillin treated mothers. Moreover, in mice born from antibiotic treated mothers the significant lymphocytosis in the peripheral blood was assessed. It was accompanied by an increase of granulocyte number in offsprings from ampicillin treated mothers and increase of monocyte number in those of cloxacillin treated. In conclusion, it could be suggested that ampicillin treatment during pregnancy would exert some effect on the development of lymphopoietic system of fetuses.

Differential effects of experimental hyperlipidemia on various types of rabbit peripheral blood lymphocyte receptors.

Rabbits fed a high-cholesterol diet for 3 months showed an increase in peripheral blood lymphocytes with membrane receptors for the Fc portion of immunoglobulin G and a decrease in lymphocytes with receptors for the Fc portion of immunoglobulin M and also with receptors for the third component of the complement. Thus our results indicate changes in lymphocyte membrane receptor expression in experimental hyperlipidemia that may supposedly influence some lymphocyte functions.

Immunomodulatory action of ampicillin.

Ampicillin was found to have an immunoenhancing effect on the expression of FcM human lymphocyte receptors, on the angiogenic potential of human lymphocytes and on the production of anti-SRBC antibody after in vivo administration to low-responder strains of mice. The effect of ampicillin on the graft-versus-host disease in irradiated F1 recipients of spleen cells from presensitized donors was found to be stimulatory when the drug was given to the recipients and suppressive when the drug was administered to the donors which had been preimmunized with a lower dose of antigen.

Ampicillin effect on lymphatic tissue of mice and its reaction to sheep erythrocytes.

Ampicillin given to B6A F1 mice in multiple doses of 250 mg/kg of body weight caused changes of the thymus with reduction in size, weight and cellularity of the organ, and with a decrease of the migratory activity in vitro of thymocytes derived from mice treated with ampicillin. Ampicillin produced also a stimulatory influence on the reaction of B-lymphocyte zone in the subpopliteal lymph nodes of mice stimulated with sheep erythrocytes, and delayed the development of this reaction in the thymus-dependent zone of these nodes in response to syngeneic erythrocytes.

Immunosuppressive effect of concanavalin A. IV. The effect of Con A on the local graft versus host (GvH) reaction in the mouse popliteal lymph node.

In the applied parental F1 hybrid system, the GvH character of the regional lymph node reaction was checked against syngeneic and the absolute control systems. It was found that a cortical B-cell zone of the recipients did not participate in the acute phase of the GvH reaction. Cytologically, the GvH response consisted in a significant increase in large pyroninophilic cells, histologically - in a cellular proliferation and pyroninophilia, as well as in a marked angiogenesis beyond the cortical zone of the lymph node. The injection of Con A into the F1 hybrid mice, 24 hours before the administration of the parental cells, inhibited the earlier (presumably donor origin) cellular reaction and intensified the later (presumably host origin) response.

The effect of ampicillin on skin graft survival and antibody response in B6A F1 mice.

The effect of ampicillin on the immune response to sheep red blood cells (SRBC) and on the survival of BALB/c skin allograft in B6A F1 mice was studied. At the non-toxic doses of 250-500 mg/kg per day, given repeatedly before or after injection of SRBC or skin grafting, there was a marked enhancement of the antibody response and a prolongation of skin graft survival from 12.7 +/- 0.23 days in untreated mice to 13.7 +/- 0.19 days and 15.2 +/- 0.75 days in mice treated before or after skin grafting, respectively.

The immunosuppressive effect of concanvalin A. III. Influence on the survival time of skin allografts in mice.

The influence of Con-A on the survival time of skin grafts in mice was examined. F1 mice (BALB/c X DBA/2) were the skin donors and C57BL X C3H were the recipients. More than a two-fold prolongation in the survival time of the grafted skin was obtained by a single 100 microgram dose of Con-A given intravenously to mice 24 h prior to grafting. Con-A given in a similar manner three days after grafting did not influence the skin graft. The results obtained substantiate our earlier observations which suggested that Con-A administered prior to an antigen may inhibit the immunological response.

The effect of ATG treatment on LyMIF and LyMSF production in mixed lymphocyte cultures of rabbits after renal allotransplantation.

Rabbit lymphocytes, settled up in microcappillary tubes migrate into culture medium. In the previous communication we have shown that migration of mixture of lymphocytes derived from two blood donors was significantly inferior in 73% of cultures as compared to the corresponding monocultures. 4% of mixed cultures exhibited significant enhancement of migration and in 23% no effects were seen. Use of lymphocytes from rabbits 4-6 days after kidney allotransplantation and their corresponding donors resulted in conversion of this ratio. Percentage of cultures exhibiting enhancement of migration (production LyMSF) rose to 39, 46% of cultures were inhibited (produced LyMIF) and 15% did not show any significant effect. Treatment of kidney recipients with ATG exerted strong inhibitory effect on the appearance of LyMSF and on the appearance of LyMIF in mixed cultures. These results suggested that LyMIF and LyMSF were produced by separate cell populations.