Cytotoxic Activity Of Splenocytes Research Articles

Mycobacterial surface characters remodeled by growth conditions drive different tumor-infiltrating cells and systemic IFN-γ/IL-17 release in bladder cancer treatment

ABSTRACT The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-ɣ, and bladder infiltration of selected immune cells such as ILCs and CD4TEM. BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3+ (CD4+ and CD8+) T cells, together with high systemic IFN-ɣ release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions.

Selection of the most immunogenic antigen for DNA vaccination against neuroblastoma

Evaluation of the antigen immunogenicity is one of the initial steps in selecting an effective target for antitumor vaccination. This is most relevant for tumors that have no specific markers or have a weak mutational status like neuroblastoma. The purpose of this study is to select the most immunogenic antigen for DNA vaccination against neuroblastoma and to conduct a comparative analysis of predictive programs and a mouse model. To assess the immunogenicity C57Bl/6 female mice 8-10 weeks of age (n=25) were used. The mice received two vaccinations with a DNA construct based on tyrosine hydroxylase, Birc5, and Phox2b antigens. On the 28th day, the spleens were removed and the cytotoxic activity of splenocytes and the level of production ofIFNγ were assessed. The highest levels of cytotoxic activity (18.4%) and IFNγ (60% of those responding to stimulation) were observed after vaccination with Phox2b. Thus, the most immunogenic antigen according to IEDB showed the best results in the mouse model and can be further used for DNA vaccination in neuroblastoma.

Allogeneic tumor cell line-based vaccines: A good alternative to autologous and cancer stem cell vaccines in colorectal cancer.

Objective(s):Besides the uncertainty about colorectal cancer stem cell (CCSC) markers, isolating, purifying, and enriching CCSCs to produce CCSC vaccines is highly challenging. However, allogeneic vaccines developed from CRC cell lines can provide universal, comprehensive, inexpensive, simple, and fast approach to cancer treatment. Materials and Methods:CCSCs were isolated from human CRC tissue using the in vitro sphere formation assay and then characterized through gene expression analysis, in vivo and in vitro tumor formation assay, karyotyping, and surface marker detection. Subsequently, CCSCs and two CRC cell lines (HT-29 and SW-480) were inactivated with cisplatin (CDDP) and administrated as vaccines to the three groups of athymic C57BL/6 nude mice. Afterward, tumorigenesis was challenged with HT-29 cells. The antitumor effect of vaccines was evaluated by tumor and spleen examination and immune response analysis. The cytotoxic activity of splenocytes and serum levels of TGF-β and IFN-γ were measured by Calcein-AM cytotoxicity assay and enzyme-linked immunosorbent assay (ELISA), respectively.Results:The results of gene expression analysis showed that CCSCs are CD44+CD133-LGR5-. All vaccinations resulted in decreased tumor growth, spleen enlargement, enhanced serum level of IFN-γ and TGF-β, and increased cytotoxic activity of natural killer (NK) cells. The antitumor efficacy of the CCSC vaccine was not more than CRC cell line-based vaccines. Interestingly, the allogeneic SW-480 vaccine could effectively inhibit tumorigenesis.Conclusion:Despite the great challenge in developing CCSC vaccines, allogeneic vaccines based on CRC cell lines can efficiently induce antitumor immunity in CRC.

A Sensitive and Simple Method to Assess NK Cell Activity by RT-qPCR for Granzyme B Using Spleen and Blood

A new assay method for natural killer (NK) cell activity was established using quantitative RT-PCR (RT-qPCR) to determine the gene expression of granzyme B (GzmB) and perforin 1 (Prf1). The RT-qPCR method was compared to a conventional cytotoxic assay. Upregulated expression of GzmB and Prf1 mRNA and enhanced cytotoxic activity were observed in splenocytes from lipopolysaccharide (LPS)-treated rats. A high correlation, R2 = 0.71, was observed between the gene expression of GzmB and the cytotoxic activity of splenocytes from these rats, indicating that GzmB RT-qPCR is a reliable alternative method to assess NK cell activity/activation. Remarkably, 12.6- to 59.7-fold upregulation of GzmB mRNA expression was observed in leukocytes, the spleen, and splenocytes from LPS-injected rats. Its upregulation appeared to be dose-dependent on the LPS concentration in the range of 0.01 - 0.1 mg/kg. Whereas, only 1.3- to 1.9-fold increase of cytotoxic activity was detected in splenocytes from the rats treated with LPS in the same range. From these, it is evident that, to assess NK cell activity/activation, the GzmB RT-qPCR method is highly sensitive compared with the conventional cytological assay. Furthermore, this GzmB RT-qPCR method is advantageous, as it does not require freshly prepared splenocytes and cell culture procedures. The convenience of GzmB RT-qPCR enables the use of whole blood, leukocytes, the spleen, and/or their frozen samples to evaluate NK cell activity/activation.

FEATURES OF IMMUNE SYSTEM DISORDERS IN EXPERIMENTAL BRAIN TRAUMA IN RATS

Introduction. Traumatic brain injury (TBI) is one of the common diseases of the person, which is accompanied by high mortality and disability of the victims. In the pathogenesis of TBI there are at least 2 periods that are associated with both primary nerve cell injury by the traumatic factor and secondary inflammatory-destructive changes that develop over a long period after the injury. An important role in the development of the second period of TBI is played by the body’s immune system, which can complicate the course of TBI and act differently depending on the severity of the injury.
 The goal of the work. The work investigated the state of proliferative and cytotoxic ability of splenocytes in light TBI in rats, which was caused by a drop in weight of 50 g from a height of 120 cm per animal.
 Materials and results. Studies have shown that within 24 hours after injury, there is an increase in the proliferative activity of splenocytes in the test for proliferation with mitogens, especially with KonA mitogen. While the cytotoxic activity of splenocytes is significantly inhibited at this time and the number of hyperdiploid cells in the spleen is reduced. At a later date after the injury, for 5 days, there is a significant recovery of immunological parameters, indicating that from the first hours after a mild TBI, the immune system changes in different directions, mainly towards the activation of proliferation, which may complicate the course the traumatic period.
 Conclusions. In experimental mild TBI, rats have differentiated changes in the activity of immune cells, which indicate the activation of the immune system in the early stages after injury.

The effect of perioperative analgesic drugs omnopon and dexketoprofen on the functional activity of immune cells in murine model of tumor surgery.

We aimed to investigate the effect of perioperative analgesia with nonselective cyclooxygenase-2 inhibitor dexketoprofen and opioid drug omnopon on the functional activity of immune cells in tumor excision murine model. Lewis lung carcinoma cells were transplanted into hind paw of C57/black mice. On the 23th day tumor was removed. Analgesic drugs were injected 30 min before and once a day for 3 days after the surgery. Biological material was obtained a day before, 1 day and 3 days after the tumor removal. IFN-γ, IL-4, IL-10 and TGF-β mRNA levels in splenic cells were assessed by quantitative real-time RT-PCR. Cytotoxic activity of splenocytes was estimated by flow cytometry. We found that in splenocytes of mice received opioid analgesia IL-10 mRNA level was increased 2.3 times on day one after the surgery compared to preoperative level (P < 0.05), while in dexketoprofen group this parameter did not change. IFN-γ gene expression level on day 3 after tumor removal was 40% higher in splenocytes of dexketoprofen treated mice as compared with omnopon treated animals (P < 0.05). Cytotoxic activity of splenocytes on day 3 postsurgery was (62.2 ± 2.4)% in dexketoprofen against (50.2 ± 3.3)% in omnopon group. In conclusion, perioperative analgesia with cyclooxygenase inhibitor dexketoprofen in contrast to opioid analgesia with omnopon preserves higher functional activity of murine immune cells in the experimental model of tumor surgery.

Inhibitory and immunological effects induced by the combination of photodynamic therapy and dendritic cells on mouse transplanted hepatoma

ObjectiveTo investigate the anti-tumor and immune efficacy of photodynamic immune-therapy (PIT), the combination of photodynamic therapy and dendritic cells (DC), on murine Heps hepatoma. MethodsDCs were derived from syngeneic mouse bone marrow and then labeled with DAPI in vitro. The hepatoma model was established by subcutaneous inoculation with Heps cells in one hundred and twenty-eight mice. They were then divided into four groups at random: control group, PDT group, DC group and PIT group. Tumors in the control group were injected with normal saline. Mice in the PDT group were injected with the photosensitizer Deuteporfin 24 h before irradiation. Mice in the DC group were injected with DAPI labeled dendritic cells intratumorally. Mice in the PIT group were further given an injection of DCs after photoirradiation. Tumor growth and survival time were recorded after treatment. Fluorescence of tumor draining lymph nodes was evaluated under fluorescence microscope. Cytotoxic activity of splenocytes was tested by standard lactate dehydrogenase (lactate dehydrogenase, LDH) release assay. Results(1) Tumor growth was significantly slowed down in the PDT and PIT group compared to the control group (P<0.01). (2) The mean survival time was significantly prolonged in the PDT and PIT group. (3) The number of fluorescent cells in the draining lymph nodes from DC group was higher than that of the PIT group. (4) The anti-tumor activity of splenocytes in the PDT and PIT group was significantly higher than that of the DC and control groups (P<0.01, P<0.01). ConclusionsThe present study suggests that PDT can inhibit tumor growth and induce anti-tumour immune response. The combination of PDT induced by Deuteporfiin and dendritic cell is capable of amplifying the antitumor immune response.

CYTOTOXIC ACTIVITY OF IMMUNE CELLS FOLLOWING ADMINISTRATION OF XENOGENEIC CANCER VACCINE IN MICE WITH MELANOMA B-16

To study the effects of xenogeneic cancer vaccine (XCV) developed on the basis of nervous tissue antigen from rat embryo of late gestation period and protein-containing metabolite of Bacillus subtilis with molecular weight of 70 kDa, on specific and unspecific antitumor reactions of cellular and humoral chains of immune system, and to analyze possible mechanisms of its antimetastatic action. XCV was administered triply with 3-day intervals after surgical removal of experimental melanoma В-16 in C57Bl/6 mice. Cytotoxic activity (CTA) of splenocytes against target cells К-562 as well as CTA of splenocytes, peritoneal macrophages (PM) and blood serum against melanoma В-16 target cells were determined using МТТ test. The content of circulating immune complexes (CIC) in blood serum was evaluated by precipitation reaction. Immunologic effects of XCV vaccination in experimental animals with surgically removed melanoma B-16 in comparison with similarly treated unvaccinated mice were as follows: prevention of medium molecular weight CIC accumulation in blood serum during all observation period, significant increase (р < 0.05) of CTA of effectors of unspecific antitumor immunity (natural killer cells - NK - by 25.5 ± 1.7 vs 12.5 ± 5.4%, and PM - by 37.3 ± 0.6 vs 32.0 ± 0.9%, respectively) at 37(th) day after the surgery, and also preservation of functional activity of specific cytotoxic lymphocytes at the level of intact control. The results of the study allow propose that antimetastatic effect of XCV vaccination could be based on increased CTA of NK and PM, and preservation of CTL functional activity at late terms after surgical removal of B-16 primary tumors.

Effect of sulfated polysaccharides from brown seaweed Laminaria japonica on the morfology of lymfoid organs and functional characteristics of immunocompetent cells

The effect of sulfated polysaccharide fucoidan from the brown alga Laminaria japonica on morphological characteristics of mouse lymphoid organs, subpopulations of spleen mononuclear leukocytes, cytokine production and cytotoxic activity of splenocytes has been investigated. Fucoidan promoted activation and proliferation of lymphoid hematopoietic cells in primary and secondary immunogenesis bodies, increased expression of markers CD19, NK, NKT, CD25, MHC II, TCR, TLR2 and TLR4, the cytotoxic activity of splenocytes and production of immunoregulatory and proinflammatory cytokines (IL- 2, IL-12, IFN-g, TNF-a, IL-6). This suggests activation of effector mechanisms of innate immunity and adaptive immune responses via the Th-1 type.

Reconstructed Adeno-Associated Virus with the Extracellular Domain of Murine PD-1 Induces Antitumor Immunity

The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

Shikonin induces immunogenic cell death in tumor cells and enhances dendritic cell-based cancer vaccine

Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.

Immunization with DNA Vaccine Expressing Herpes Simplex Virus Type 1 gD and IL-21 Protects against Mouse Herpes Keratitis

The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. In this study, we developed a DNA vaccine expressing HSV-1 glycoprotein D (gD) and mouse interleukin-21(IL-21) and intramuscularly inoculated mice 3 times at 2-week intervals with a total of 300 μg/mouse. Two weeks after the last immunization the specific antibody, splenocyte proliferative response to gD, IFN-γ and IL-4 as well as the cytotoxic activities of splenocytes and natural killer (NK) cells were assayed. Immune protection against herpes keratitis was concurrently evaluated in the immunized mice after HSV-1 challenge of the mouse cornea. The results showed that the DNA vaccine pRSC-gD-IL-21 generated higher levels of antibody, IFN-γ and IL-4, and enhanced the splenocyte proliferative response to gD as well as the cytotoxic activity of splenocytes and NK cells to target cells compared with the response in either the pRSC-gD or mock plasmid pRSC immunized mice. Importantly, the pRSC-gD-IL-21 ameliorated herpes keratitis severity and time course after corneal infection with HSV-1. The findings suggest that the DNA vaccine pRSC-gD-IL-21 may induce an immune response that can limit HSV-1 infection and development of herpes keratitis in the immunized mice.

Interleukin-21 Activates Cytotoxic T Lymphocytes and Natural Killer Cells to Generate Antitumor Response in Mouse Renal Cell Carcinoma

We evaluated the antitumor effects of IL-21 gene transfer into mouse RenCa renal cell carcinoma cells, so that cells could spontaneously secrete IL-21. We also investigated the mechanisms underlying this antitumor effect. The IL-21 gene was introduced into RenCa cells by the liposome mediated method using Lipofectamine. The in vivo antitumor effect of IL-21 secreting RenCa cells was assessed by subcutaneous injection into syngeneic BALB/c mice. Mechanisms underlying the antitumor effects were investigated in syngeneic mice in which CD8 T, CD4 T or natural killer cells had been depleted using the corresponding antibody. The cytotoxic activity of splenocytes in mice injected with IL-21 secreting RenCa cells was determined using the CytoTox 96 nonradioactive cytotoxicity assay. Immunohistochemical examinations were performed to investigate infiltrating cells around tumor sites in vivo. Tumor vaccine study was also performed. IL-21 secreting RenCa cells were almost all rejected following subcutaneous injection into syngeneic mice. The antitumor effect of IL-21 secreting RenCa cells remained in mice in which CD4 T cells had been depleted but it was totally abrogated in mice depleted of CD8 T cells or natural killer cells. Cytotoxic activities of splenocytes were higher in IL-21 secreting RenCa cell rejected mice than in parental RenCa mice. Immunohistochemical study also supported the involvement of CD8 T cells and natural killer cells in the antitumor effect of IL-21 secreting RenCa cells. Moreover, mitomycin C treated IL-21 secreting RenCa cells inhibited the growth of parental RenCa at distant site. IL-21 secreting RenCa could be rejected in syngeneic mice by the activation of CD8 T cells and natural killer cells. Moreover, mitomycin C treated IL-21 secreting RenCa cells could work as a tumor vaccine for parental RenCa.

Evaluation of anti-tumor effects of tumor cell lysate enriched by HSP-70 against fibrosarcoma tumor in BALB/c mice

The cytosolic members of the heat shock protein 70 (HSP-70) family have been shown to elicit protective cell mediated immunity in animal tumor models. The aim of this study was to investigate the effect of the HSP-70 enriched lysate of heated tumor cells as vaccines in cancer immunotherapy in the mouse model for WEHI-164 fibrosarcoma. Three animal bearing tumor groups were investigated: test group; vaccinated with enriched HSP-70 tumor lysate, control group I; vaccinated with tumor lysate only and control group II; received PBS. The results indicated that vaccinated mice in the test group had resulted in a significant reduction in tumor size and longer survival. To find the mechanism of these results, we measured the splenocytes proliferation, tumor infiltrated lymphocytes and cytotoxic activity of the splenocytes. The results indicated a significant increase in the proliferation of mouse splenocytes, a significant increase in the CD8+ lymphocytes as well as significant increase in the cytotoxic activity of splenocytes against the target cells in the test group. In addition, we analyzed the shifting of Th1/Th2 in all the groups. The results indicated a significant increase in the IFN-γ production in the test group. These findings provided a useful therapeutic model for development of approaches to cancer treatments.

The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)-based immunotherapy. Interleukin 12 (IL-12) and granulocyte macrophage colony-stimulating factor (GM-CSF) play a pivotal role in inducing Th1 and cytotoxic T lymphocyte (CTL) responses. In this study, DCs expressing the natural tumor antigen gp70 of BALB/c-derived CT26 were adenovirally transduced with the IL-12 gene and/or GM-CSF gene, and it was examined whether vaccinations using these genetically engineered DCs can induce strong therapeutic antitumor immunity. Mice were immunized once by subcutaneous (s.c.) injection with genetically modified DCs. The cytotoxic activity of splenocytes against CT26 was assayed in a 51Cr-release assay 14 days after immunization. The therapeutic efficacy of the vaccination was examined in s.c. tumor models. The cytotoxic activity of CTLs against CT26 in mice immunized with DCs expressing gp70 (DC-AxCAgp70) was significantly augmented by co-transduction with the GM-CSF/IL-12 gene (p<0.0001) and remarkably reduced by the depletion of CD4+ or CD8+ cells (p<0.01). The cytotoxic activity against CT26 of the plain spleen cells in mice immunized with DC-AxCAgp70/GM-CSF/IL-12 was significantly higher than that in mice immunized with DC-AxCAgp70 (p<0.0001), and this activity decreased to almost 50% upon the depletion of NK cells. Vaccinations using DC-AxCAgp70/GM-CSF/IL-12 or DC-AxCAgp70/IL-12 could elicit potent therapeutic immunity in s.c. tumor models; tumor-free mice were observed in these vaccination groups. However, there was no significant difference between these two groups. A vaccination therapy using DCs co-transduced with the TAA gene and Th 1-type cytokine genes, especially the IL-12 gene, is ideal for immunotherapy in terms of the activation of DCs, NK cells, CD4+ T cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.

Suppression of Natural Killer Cell Activity on Candida Stellatoidea by a 50 Hz Magnetic Field

Exposure to electromagnetic fields (EMF) is ubiquitous for almost all individuals living in industrialized countries. Epidemiological and laboratory studies suggest that exposure to Extremely Low Frequency (ELF) EMF increase cancer risk. The immune system functions as one of the body's main protective mechanisms, and Natural Killer (NK) cells are a subset of lymphocytes that can destroy several types of tumor cells. In this study, we investigated, NK cell activity after exposure to a 50 Hertz (Hz), 2 mT magnetic field generated by a Helmholtz Coil. Nineteen male, 10–12 week old guinea pigs were used, and NK cytotoxic activity of splenocytes was measured in vitro by natural anticandidial colorimetric index. The Mann–Whitney U test was applied for statistical analysis. NK cell cytotoxic activity was decreased in exposed compared to controls. Our data suggests that part of the immune system, the NK cell, can be suppressed by a 50 Hz magnetic field.

Pregnancy-induced antitumor cytotoxicity of T cell-rich fraction against mammary adenocarcinoma cells in rats.

Our earlier observations indicated that splenocytes from parous rats have high cytotoxic activity against mammary tumor cells in vitro. It has also been observed that this cytotoxic activity of splenocytes from parous rats can be adoptively transferred to virgin rats of same age. The present investigation is an attempt to determine the cell type in spleens of parous rats that cause the cytotoxicity against mammary tumors. The spleens from both virgin and parous rats were removed aseptically and T and B cell-rich fractions were separated. 7,12-Dimethylbenz[a]anthracene-induced rat mammary tumor epithelial cells were used as targets and the T and B cell-rich fractions from either virgin or parous rats were used as effectors. The parous rats were divided into two groups according to the time period after parturition. Group I contained rats that were 5-13 days after delivery, while the rats in group II were 14 or more days post parturition. In vitro cytotoxicity was determined by incubating the tumor cells with spleen cells in the target/effector ratio of 1:3, 1:10 and 1:30 and using the standard 51Cr release assay. In all ratio groups of T cell-rich fractions particularly in group II, there was significantly higher cytotoxicity against mammary tumor cells. None of the B cell-rich fractions from parous rats were significantly more cytotoxic than those from virgin controls.

Immunostimulation by a partially modified retro-inverso-tuftsin analog containing Thr1.sum..psi.[NHCO](R,S)Lys2 modification

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

Kinetics of Cell-Mediated Cytotoxicity in Mice Fed Diets of Various Fat Contents

We previously reported lower mitogen-induced blastogenic and cytotoxic activity of splenocytes from C3H/OUJ female mice fed 20% soybean oil (SBO) in their diet compared to those fed 5% SBO. The present study examined the kinetics of cell-mediated cytotoxicity using the same animal model and dietary treatments. Kinetic parameters were determined by analyzing the lytic efficiency of splenocytes cultured for various times with several concentrations of radiolabeled P815 mastocytoma cells. The apparent avidity constant (K1/2) of the reaction was not changed by dietary SBO intake (1.0 +/- 0.2 x 10(5) cells for 20% SBO versus 1.3 +/- 0.3 x 10(5) cells for 5% SBO). However, the maximum velocity (Vmax) of the reaction for splenocytes from mice fed 20% SBO was significantly lower than that for splenocytes from mice fed 5% SBO (1.4 +/- 0.2 x 10(4) cells/h for 20% SBO versus 2.3 +/- 0.4 x 10(4) cells/h for 5% SBO, p less than 0.05). The evidence indicates that the rate of target cell lysis, but not the avidity of the lymphocytes for the target cell antigen, was altered by increasing dietary SBO concentration.

Induction of candidicidal activity in mice by immunization withCandida albicansribosomes

Mice immunized with ribosomes from Candida albicans are protected against experimental systemic candidiasis. In this study we investigated the candidacidal activity of spleen cells from immunized animals as measured by 51Cr release from pre-labelled yeast cells. It was found that the anti-candidal cytotoxic activity of splenocytes from immunized mice was significantly higher than that of spleen cells from non-immunized controls with various effector to target (E:T) ratios, but optimal results were obtained with an E:T ratio of 10:1. The cytotoxic activity of splenocytes as measured by the 51Cr release assay correlated well with the capacity of the cells to inhibit candidal growth as determined by quantitative plating. This candidacidal activity was not antibody dependent but increased killing was obtained by adding fresh (but not heat inactivated) mouse serum. The enhanced candidicidal activity was inhibited by removal of plastic- or nylon-adherent cells from the cell suspension but not by treatment with anti-Thy 1.2 serum and complement. The data indicate that a candidacidal cell population is induced in the spleens of animals immunized with C. albicans ribosomes.