Humoral stimulating activities in post-cyclophosphamide rat sera and their purified fractions.

Enhanced humoral, stimulating activities (HSAs) of post-cyclophosphamide (CY) sera and their purified fractions, acting on mitogen activated T-lymphocytes, were detected in Wistar rats after treatment by high single doses of the aplasia producing alkylating cytostatic drug CY. These activities, monitored in vitro, were partially purified from post-CY sera, collected 2, 4, and 7 days after treatment, yielding fractions with higher humoral stimulating activity. The preliminary purification included molecular cutting by Amicon-Diaflo filters (1-30 kDa pore size range) and gel-filtration on Sephadex G-50 and G-75. Results show that post-CY sera partially purified fraction (10-20 kDa), enhances the proliferation of hydrocortisone resistant (HCR) T-lymphocytes up to threefold under microculture conditions (P << 0.001); partially purified post-CY sera fraction (10-20 kDa) increases the proliferative response of T-cells in microcultures to the mitogenic 145-2C11 monoclonal antibody (mAb) up to tenfold (P << 0.001). These results show that the activity of stimulating factors is localized in the molecular weight range of 10-20 kDa.

A laser nephelometric method for measuring alternative pathway complement activity of human and murine serum.

A kinetic method for the evaluation of alternative pathway complement activity in man and mice is presented. A laser nephelometer was employed for detection of non-sensitized rabbit erythrocyte lysis based on the observation that the intensity of laser scatter (LS) is proportional to the number of erythrocytes in suspension. During erythrocyte lysis a continuous decline in LS is observed since lytic products do not evoke LS. Utilizing the indirect Coombs test and cross-electrophoresis it was shown that rabbit erythrocyte lysis causes activation of the alternative complement pathway. This method is modified for room temperature, is independent of sample hemoglobin content and, in its micro version, it can be done with 10 microliter of human serum, i.e. 50 microliters of murine serum.

Antibodies against rabbit red blood cells in murine serum and their effect on the activity of complement alternative pathway.

Antibody content against rabbit red blood cells (anti-RaRBC) in murine sera of different strains (Swiss, CBA, C57BL/6, AKR, BALB/c) and activity of complement alternative pathway (AP) were investigated. In contrast to the CBA and C57BL/6, random-bred Swiss strain and inbred BALB/c and AKR strains are good producers of these natural antibodies. There is no correlation between AP activity and anti-RaRBC content. Isolated human anti-RaRBC antibodies, IgM and IgG classes, lead to the enhancement of APhu and APmo activity, contrary to the murine anti-RaRBC which belong solely to IgM class, and do not express this capability.

Interspecies differences in expression of cytokeratin polypeptides within thymic epithelium: a comparative immunohistochemical study.

Cytokeratin (CK) polypeptide expression within the thymic epithelium of several mammalian species (mouse, rat, calf, pig, rabbit, and human) has been analyzed by the streptavidin-biotin immunoperoxidase method. Comparative analysis by a large panel of 17 monoclonal antibodies (mAbs) specific for individual CK polypeptides, pairs, or groups showed considerable heterogeneity of thymic epithelial cells (TEC) in each species. In addition, extreme interspecies difference in CK contents was observed. Four main phenotypic zones: the subcapsule/perivascular area, cortex, medulla, and Hassall's corpuscles (HC) were clearly identified, each characterized by different CK expression. Medullary TEC were more heterogenous and shared common CK polypeptides either with subcapsular/perivascular TEC, cortical TEC, or HC, in most species.

Aberrant levels of cytokines within the healing wound after burn injury.

OBJECTIVE: To study the effect of a burn injury on the course of cellular and cytokine changes in a wound and the relationship of these cytokines to the amounts of protein and collagen deposited at the site of the wound. DESIGN: A randomized control trial was done in which one group of rats were subjected to a severe burn injury. With the use of a sponge matrix model, the wound-healing parameters were evaluated. MATERIALS: A random sample of eight inbred albino Oxford rats per group were used in all experiments. INTERVENTIONS: Rats were subjected to a severe scald injury. Polyvinyl sponges were used as the wound-healing model. MAIN OUTCOME MEASURE: The obtained results implied that the wound-healing process is impaired after a severe burn injury. RESULTS: The wounds in these animals with burn injuries contained a lower number and an altered type of infiltrating cells with aberrant levels of cytokines, higher levels of interleukin-6, and lower levels of tumor necrosis factor and interleukin-1 in the fluids of the wounds. The parameters of healing (amounts of protein and collagen deposited at the site of the wound) were significantly lower in animals with burn injuries on days 7 and 14. CONCLUSION: The underlying mechanism of the impaired healing of a wound after burn injury could lie in the altered migration of inflammatory cells to the site of the wound and in the aberrant cytokine levels within the wound.

Interleukin-1 in vivo modulates trauma-induced immunosuppression.

To elucidate the mechanisms underlying trauma-induced immunosuppression and decreased IL-2 production we evaluated: 1) the effect of trauma on IL-1 production at different time intervals and 2) the effect of IL-1 in vivo administration on immune functions (IL-1 production, IL2 production, NK cell cytotoxicity) in normal and traumatized mice. Experiments were performed on CBA/H mice a) subjected to scald injury (sacrificed 3 h and 6 h later) b) treated with IL-1 in vivo (human recombinant IL-1 beta 100 ng/mouse, sacrificed 21 h and 24 h later) and c) subjected to both IL-1 in vivo treatment and scald (IL-1 was given 18 h before scald, animals were sacrificed 3 h and 6 h after scald i.e. 21 h and 24 h after IL-1 administration). Our results demonstrate that trauma alone increases IL-1 production from 1 h to 24 h after trauma infliction. Recombinant IL-1 given in vivo also induces a significant rise in IL-1 production. When mice were subjected to both trauma and IL-1 in vivo treatment, the rise in IL-1 production was not additive. Trauma induced severe depression of IL-2 production which could not be overcome by in vitro addition of IL-1 to IL-2 producing splenocytes from traumatized mice. In contrast, IL-1 administered in vivo stimulated IL-2 production in normal mice, and when given prior to trauma infliction, it completely abrogated trauma-induced suppression of IL-2 production. The same effect was seen on NK cell cytotoxicity (an IL-2 dependent function).(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct spectrum of N-ras mutations in aml and mds patients in yugoslavia.

Mutations that activate ras genes were demonstrated to be associated with certain types of malignancies. Multiple point mutations were predominantly found in the N-ras and occasionally in the K-ras genes. The analysis of 4 MDS, 23 AML and 11 CML patients from Yugoslavia revealed the prevalence of the N-ras mutation (83%) over K-ras mutations (17%). Although the frequencies of the N- and K-ras mutations in these patients were similar to the ones reported for patients from USA and Japan, the N-ras mutational spectra considerably differed. The prevailing type of mutation in patients from Yugoslavia was G-to-T transversion at the first position in the codon 12 of the N-ras gene. This study supports a hypothesis that different geographical and environmental factors may cause the accumulation of different type of point mutations in the same target gene.

Disease modifying and immunomodulatory effects of high dose 1 alpha (OH) D3 in rheumatoid arthritis patients.

OBJECTIVE: Vitamin D analogues such as 1 alpha (OH) D3 (alphacalcidiol) have a possible physiological paracrine effect on cell proliferation and differentiation. Experimentally established possibilities to prevent autoimmune diseases suggest that alphacalcidiol may have therapeutic value as an immunomodulatory agent in patients with rheumatoid arthritis. METHODS: We organized a 3-month open-label trial on 19 patients being treated with standard DMARD therapy for acute RA. They were divided into 2 subgroups, those with highly active RA and those with moderately active RA. Their regular drug regimen was maintained during the trial and oral alphacalcidiol 2 micrograms/day was added. Therapy results were evaluated by ESR, CRP, morning stiffness, the Richie index, and the Lee index. Immunomodulatory effects were investigated by measuring lymphocyte proliferation and apoptosis both in the patients and in vitro in 10 nM alphacalcidiol-supplemented culture medium. RESULTS: After 3 months, high dose oral alphacalcidiol therapy showed a positive effect on disease activity in 89% of the patients (45% or 9 pts. with complete remission and 44% or 8 pts. with a satisfactory effect). Only two patients (11%) showed no improvement, but no new symptoms occurred. No side effects were observed. CONCLUSION: These results suggest that alphacalcidiol is a powerful immunomodulatory agent with fairly low hypercalcemic activity. Clinical improvement was strongly correlated with the immunomodulating potential of this agent. We noticed dual effects on lymphocyte proliferation and apoptosis according to the prior cell activation state. Alphacalcidiol could therefore possibly be used as an adjunct therapy with DMARDs in patients with rheumatoid arthritis.

Thymic response to thermal injury in mice: I. Alterations of thymocyte subsets studied by flow cytometry and immunohistochemistry.

The dynamics of thymocyte subset changes in mice subjected to sublethal thermal injury were studied in cell suspensions by flow cytometry and in situ by immunohistochemistry. Thermal injury caused acute thymic involution in the first 2 days which was the consequence of a considerable decrease in numbers of Thyl.2high+ CD4+ CD8+, cortical thymocytes. Medullary, Thyl.2low+ thymocytes were more resistant and their relative values increased. In the regenerative phase (2-14 days) the recovery of large CD4- CD8-, early thymocytes, mainly localized in the subcapsular area of the thymus, preceded the regeneration thymocytes of the cortical phenotype. Judged by the absolute numbers of medullary thymocytes it can be seen that CD4+ CD8- (T-helper/inducer cells) were more sensitive to the effect of thermal injury than CD4- CD8+ (T-suppressor/cytotoxic cells). While values of CD4+ CD8- cells were constantly and progressively lower during 2 weeks after thermal injury, absolute numbers of CD4- CD8+ cells showed cyclic changes with lower and higher values compared to controls. An increase in the numbers of CD4- CD8+ cells was found at day 6 after thermal injury.

Post-traumatic activation of draining lymph node cells. II. Proliferative and phenotypic characteristics.

Proliferative and phenotypic characteristics of cells in regional lymph nodes that drain burn injury were examined in rats on day 3 postburn, i.e. at the time of maximal spontaneous proliferation and of interleukin-2 and accessory cytokine (IL-1 and IL-6) production. The importance of IL-1 in spontaneous proliferation of draining lymph node cells was demonstrated by stimulation of IL-2-driven proliferation by recombinant IL-1 in vitro and by susceptibility of unstimulated proliferation to anti-IL-1 antibodies, while requirements for IL-6 in draining lymph node cell proliferation were less pronounced. Cell surface phenotyping revealed a slightly increased percentage of CD25+ cells in the blast cell population of freshly isolated draining lymph node cells after injury, which increased further during cultivation. Enrichment in CD8+ cells on day 3 following burn injury was demonstrated, while no changes in total cell population and CD4+ cells was noted. This was however preceded by pronounced percentual decrease of total T cells and CD4+ cells and by increases of B cells and MHC class II+ cells on day 1 postburn. Inhibition of draining lymph node cell proliferation by anti-MHC class II antibodies suggested that this proliferation was class II MHC dependent. The contribution of cell proliferation and/or cell influx to day 3 postburn draining lymph node cell activity is discussed.

[Immunomodulation during viral infections]. / Imunomodulacija u toku virusnih infekcija.

The paper provides a survey of immunomodulation mechanisms in the course of viral infections. The importance of viruses in the disturbance of immunological homeostasis has been pointed out. Virally induced immunomodulation can arise by: a mechanism of complete or abortive viral replication in the lymphocytes and macrophages, by soluble viral factors, by the influencing of antigen-presenting cells, and by excessive suppressor T-lymphocyte stimulation. Viral infections present a real danger to the integrity of the immunological system, and this demands a follow-up and an adequate interpretation of the arisen disorders.

Heterogeneity of rat thymic epithelium defined by monoclonal anti-keratin antibodies.

Immunohistochemical characterization of rat TEC has been studied using a panel of monoclonal anti-keratin antibodies. These mAbs identified three distinct patterns of keratin subunit expression within thymic epithelium assessed by streptavidin-biotin immunoperoxidase staining and double immunofluorescence staining. K8 and KII mAbs labelled almost all epithelium, while K18 stained cortical epithelium and a subpopulation of medullary TEC. KI, K7 and K19 mAbs bound to the subcapsular/subtrabecular flat epithelial cell layer, TEC lining some perivascular spaces in the cortex, a subpopulation of medullary TEC and interstitial epithelial cystic structures. Double immunostaining revealed further heterogeneity of subunit keratin compositions in epithelial cells of particular thymic microenvironments suggesting their different origin or development stage.

Immunohistochemical identification of six cytokeratin-defined subsets of the rat thymic epithelial cells.

Rat thymic epithelial cells (TEC) have been studied by a panel of monoclonal antibodies specific for single cytokeratin polypeptides or cytokeratin pairs. Using various combinations of single and double immunostainings 6 TEC subsets (CK types) were identified, each characterized by different cytokeratin expression. Subcapsular/perivascular TEC (TEC-CK type 1) share cytokeratins 7, 8, 19 with a subset of medullary TEC, while cortical TEC were reactive with anti-CK 8 and anti-CK 18 mAbs only (TEC-CK type 2). Additional 4 subsets were identified in the medulla; TEC-CK type 3 (CK 8 + 18 + 19 +), TEC-CK type 4 (CK 8 + 10 + 18 + 19 +), TEC-CK Type 5 (CK 8 + 10 + 10 +) and TEC-CK type 6 positive only with CK 8 of all above cytokeratins. This study extends the concept of TEC heterogeneity and might also be useful to further understanding of TEC origin, development and functions.

[Diagnostic problems of Stevens-Johnson syndrome. A case report]. / Stevens-Dzonsonov sindrom u decjem dobu. Znacaj imunoloskih parametara u dijagnostici i tumacenju patogeneze (prikaz bolesnika).

Stivens-Johnson Syndrome is a rare, severe, bullose form of erythema multiforme of unknown etiology. The role of immunological factors in its pathogenesis elucidates. A patients (Sh.V.), nine years of age, was admitted for reccurent streptococcal infections with skin and mucose membrane lesions. In June 1990 streptococcal pharyngitis, fever (38.8-39,9 degrees C) were registered. Penicillin was given. Next day bullous lesions on lips, left ear, trunk and lower extremities and vesiculose lesions with a wide, erythematose base ("iris") and then conjuctivitis were registered. Laboratory tests: SR70.; Leu - 11,0; anti-herpes Ab IgG 1/64, IgM 1/8. Stevens-Johnson was diagnosed. There was a recidivation two years after - oral lesions followed by necrosis and bleeding, after half a year a second recidivation with spreading of bullous and vesiculous lesions to penis gland with prepuce of the penis. Last recidivation in February 1993. Anamnesis: Viral meningitis in 1988. mother suffers from herpes labialis. Peripheral blood immunophenotiping lymphocite extremly indicated decreasing values of B Ly, NK and IL-2R+ cells. Bacteriological tests showed an increase of anti-Chlamidia Ab titer (IgG 1/128, IgA and IgM +). In virological testing there was no increase of titer of Abs against viral antigens (Herpes simplex virus, Varicella-Zoster virus, Citomegalovirus, Adenovirus). We conclude that Stevens-Johnson Sy to be diagnosed by characteristic clinical features, aspecialy by frequent reccurences. Immunological testing during the last recidivation showed that parameters of humoral immune reactivity were within normal ranges while revealed defects of cellular immune reactivity cannot elucidate the ethiopathogenesis of this disease.

Two novel monoclonal antibodies reactive with different components of the rat thymic epithelium.

Two novel monoclonal antibodies (mAbs) (PT10B7 and PT13D11) have been raised against molecules of rat thymic epithelial cells (TEC). Streptavidin-biotin immunoperoxidase staining and double immunofluorescence using these mAbs and anti-cytokeratin (CK) antibodies showed that PT10B7 and PT13D11 mAbs bound to different components of rat TEC. PT10B7 mAb reacted with cortical and a subset of medullary TEC, whereas PT13D11 mAb labeled subcapsular/perivascular and most medullary TEC, including TE-R 2.5 TEC line of medullary origin. Their staining patterns were different from those seen using mAbs to CK10, CK18 and CK19 polypeptides and other anti-rat TEC mAbs produced so far. The differences in immunoreactivity of these two mAbs on rat thymus during ontogeny and on other epithelial cells of adult rats were also seen. Namely, PT13D11 stained ectoderm-derived epithelia, whereas PT10B7 stained some cells of simple epithelia. Cumulatively, these results reveal a fine phenotypic heterogeneity within rat thymic epithelium.