Mesenchymal stem cell-conditioned medium prevents inflammation-induced liver and lung damage in septic mice.

AIM: Sepsis is a life-threatening condition caused by a dysregulated immune response to infection. Broad-spectrum antibiotics are used to treat it. However, due to antibiotic resistance, alternative treatments are needed. Mesenchymal stem cells (MSCs) have become a promising therapeutic tool for sepsis due to their immunomodulatory properties. The limitations of MSC therapy have led to increased attention to cell derivatives such as conditioned medium (CM). This study investigates the immunomodulatory effects of young and old MSC-CM during the inflammatory phase of sepsis. MAIN METHODS: The cecal ligation and puncture (CLP) model was used to induce sepsis in mice. The mice were divided into four groups: sham, CLP, CLP treated with young MSC-CM, and CLP treated with old MSC-CM. The CM was injected intraperitoneally at 2-, 12-, and 24-hours post-surgery. After 72 h, blood was collected and white blood cells (WBCs) were counted. In addition, serum and tissue were isolated, and the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in serum, bacterial load in the spleen, concentration of pro- and anti-inflammatory cytokines, and histopathology of liver and lung were investigated. KEY FINDINGS: MSC-CM decreased serum AST and ALT levels, bacterial load in the spleen, and pro-inflammatory cytokines in serum. In addition, tissue damage was reduced, and the survival rate and WBC count increased. There was no significant difference between the young and old MSC-CM. SIGNIFICANCE: MSC-CM effectively reduced inflammation-induced tissue damage in the liver and lungs during sepsis. Although young MSC-CM had better immunomodulatory effects than old MSC-CM, the difference was not significant.

The combination of mesenchymal stem cell- and hepatocyte-derived exosomes, along with imipenem, ameliorates inflammatory responses and liver damage in a sepsis mouse model.

Aim Sepsis is a medical emergency with no definitive treatment. Animal experiments have confirmed the therapeutic characteristics of exosomes in reducing inflammation and tissue damage. The study investigates the effect of MSC and hepatocyte-derived exosomes along with imipenem in controlling systemic and local (liver) inflammation in a mouse model of sepsis. MAIN METHODS: To induce sepsis in C57BL/6 mice, the Cecal Ligation and Puncture (CLP) model was used. The mice were given various treatments, including imipenem, MSC-derived exosomes, hepatocyte-derived exosomes, and a mixture of exosomes. Blood and liver samples were collected and analyzed for cell blood count, liver enzymes, NO levels, cytokine concentrations, and bacterial presence. The percentages of TCD3 + CD4+/CD8+ and Treg in the spleen and mesenteric lymph nodes were also assessed using flow cytometry. The pathological changes were assessed in the liver, lung, and heart tissues. In addition, the cytokine content of exosomes was measured by ELISA. KEY FINDINGS: Our results demonstrated that MSC-derived exosomes+imipenem could control systemic and local inflammation and increase the TCD4+ and Treg populations. Hepatocyte-derived exosomes+imipenem reduced inflammation in the liver and increased the TCD8+ and Treg populations. The mixture of exosomes+imipenem had the best function in reducing inflammation, maintaining all T lymphocyte populations, reducing liver damage, and ultimately increasing the survival rate. SIGNIFICANCE: The mixture of exosomes derived from MSCs and hepatocytes, along with imipenem, in the inflammatory phase of sepsis could be a promising therapeutic strategy in sepsis treatment.

The effect of mesenchymal stem cell-derived supernatant nasal administration on lung inflammation and immune response in BCG-vaccinated BALB/c mice.

Mesenchymal stem cells (MSCs) are among the known cells that can control and modulate immune responses in different circumstances, including autoimmune diseases. Also, various studies have shown that they can prevent and reduces the pulmonary inflammation caused by infectious agents. In the case of tuberculosis and inflammation caused by BCG, the granuloma has destructive effects and improper orientation of the immune response. Therefore, it is possible to prevent airway damage by preventing harmful inflammatory responses and guiding the immune system responses. This study investigates the role of nasal administration of MSCs supernatant by designing an inflammatory model in the BALB/c mice lung with BCG. MSCs are isolated from mice adipose tissue in this study and evaluated for their phenotypic and differentiation properties. After the third passage, these cells' condition medium (CM) was collected. 20 mice were divided into four groups. Group 1 receive BCG (107 CFU in 5 ml volume for 15 min) nasal administration. Group 2 treated with CM, and group 3 initially were treated with CM (in 5 ml volume for 15 min) and, after 24 h, treated with BCG nasal administration. CM treatment was continued every five days for one month. The fourth group of mice was treated with PBS nasal administration of CM and BCG. One week after the last administration, the lung tissue of mice in each group was pathologically examined. In addition, secretion of IL1-ß, IL-6, TNF-α, TGF-ß, and IL-10 in the alveolar fluid and secretion of IL-4 and IFN-γ cytokines in the supernatant of splenocytes was evaluated by ELISA. The TNF-α/IL-10 ratio in the alveolar lung fluid of the BCG received group is 2/9 and decreased to 0.58 after successive CM treatment. Therefore, it can be concluded that inflammatory responses to BCG infection in the presence of CM are balanced and pave the way for the induction of effective immune responses by reducing lung tissue damage.

Exosomal MicroRNAs as Biomarkers in Allergic Asthma.

Exosomes are extracellular vesicles that are involved in intracellular communication and different biological processes. Recently, the importance of microRNAs (miRNAs) in exosomes has been considered as biomarkers in asthma diagnosis. This study aimed to determine the expression of selective miRNAs from plasma-derived exosomes in moderate and severe asthmatic patients compared with healthy controls. Forty-six subjects including 22 patients with severe and mild to moderate allergic asthma and 24 healthy controls have entered this study. MiRNAs were extracted from the plasma exosomes and selective miRNAs (miR-21, miR-16, miR-Let7, miR-148a, miR-155, miR-125, miR-150, miR-146a, miR-223, miR-126) expressions levels were determined; using quantitative polymerase chain reaction (qPCR). In this study, we found a significant up-regulation of miR-223 and miR-21 in moderate asthmatic patients compared to the healthy controls (p=0.002, p=0.006). MiR-223 and miR-21 had the probability of 83% and 76% diagnosis estimation in moderate asthmatic patients respectively. Therefore, they could be used as biomarkers in these patients.  No expression of miR-125, miR-126, and miR-155 was found in plasma exosomes by qPCR in this study. The other miRNAs had no significant expression between different groups. Based on our findings,miR-223 and miR-21 may be considered biomarkers or used for targeted immunotherapies in asthma.

Evaluation of miR-210 expression in common variable immunodeficiency: patients with unsolved genetic defect.

BACKGROUND: Common variable immunodeficiency (CVID) is one of the most prevalent forms of primary immunodeficiency diseases (PID). CVID is characterized by failure in the final differentiation of B lymphocytes and impaired antibody production but the pathogenesis is not known in the majority of patients. We postulated that the expression pattern of miRNAs in unsolved CVID patients might be the underlying epigenetic cause of the disease. Therefore, we aimed to assess the expression of hsa-miR-210-5p and FOXP3 transcription factor in CVID cases in comparison with healthy individuals. METHODS: Eleven CVID cases with no genetic defects (all PID known genes excluded) and 10 sex and age-matched healthy individuals were enrolled in the study. T lymphocytes were purified from PBMC, and expression levels of miR-210-5p and FOXP3 mRNA were evaluated by real-time PCR. RESULTS: We demonstrated that miR-210 expression in patients was significantly higher than the control group (P = 0.03). FOXP3 expression was slightly lower in patients compared with healthy controls (P = 0.86). There was a negative correlation between miR and gene expression (r: -0.11, P = 0.73). Among various clinical complications, autoimmunity showed a considerable rate in high-miR patients (P = 0.12, 42.8%), while autoimmunity was not observed in normal miR-210 patients. CONCLUSIONS: Our results suggest a role for miR-210 in the pathogenesis of autoimmunity in CVID patients. Further studies would better elucidate epigenetic roles in CVID patients with no genetic defects.

A Systems Biology Approach for miRNA-mRNA Expression Patterns Analysis in Rheumatoid Arthritis.

OBJECTIVE: Considering the molecular complexity and heterogeneity of rheumatoid arthritis (RA), the identification of novel molecular contributors involved in RA initiation and progression using systems biology approaches will open up potential therapeutic strategies. The bioinformatics method allows the detection of associated miRNA-mRNA as both therapeutic and prognostic targets for RA. METHODS: This research used a system biology approach based on a systematic re-analysis of the RA-related microarray datasets in the NCBI Gene Expression Omnibus (GEO) database to find out deregulated miRNAs. We then studied the deregulated miRNA-mRNA using Enrichr and Molecular Signatures Database (MSigDB) to identify novel RA-related markers followed by an overview of miRNA-mRNA interaction networks and RA-related pathways. RESULTS: This research mainly focused on mRNA and miRNA interactions in all tissues and blood/serum associated with RA to obtain a comprehensive knowledge of RA. Recent systems biology approach analyzed seven independent studies and presented important RA-related deregulated miRNAs (miR-145-5p, miR-146a-5p, miR-155-5p, miR-15a-5p, miR-29c-3p, miR- 103a-3p, miR-125a-5p, miR-125b-5p, miR-218); upregulation of miR-125b is shown in the study (GSE71600). While the findings of the Enrichr showed cytokine and vitamin D receptor pathways and inflammatory pathways. Further analysis revealed a negative correlation between the vitamin D receptor (VDR) and miR-125b in RA-associated gene expression. CONCLUSION: Since vitamin D is capable of regulating the immune homeostasis and decreasing the autoimmune process through its receptor (VDR), it is regarded as a potential target for RA. According to the results obtained, a comparative correlation between negative expression of the vitamin D receptor (VDR) and miR-125b was suggested in RA. The increasing miR-125b expression would reduce the VitD uptake through its receptor.

Immunomodulatory and Therapeutic Effects of Mesenchymal Stem Cells on Organ Dysfunction in Sepsis.

ABSTRACT: Sepsis is a life-threatening disorder that is caused by a dysregulated inflammatory response during an infection. The disease mostly affects pregnant women, newborns, and patients in intensive care units. Sepsis treatment is a significant part of a country's health budgets. Delay in the therapy causes irreversible failure of various organs due to the lack of blood supply and reduction of oxygen in the tissues and eventually increased mortality. The involvement of four or five organs by sepsis has been attributed to an increased risk of death to over 90%. Although antibiotics are at the first line of sepsis treatment, they do not possess enough potency to control the disease and prevent subsequent organ failure. The immunomodulatory, anti-inflammatory, anti-apoptotic, and anti-microbial properties of mesenchymal stem cells (MSCs) have been reported in various studies. Therefore, the application of MSCs has been considered a potentially promising therapeutic strategy. In preclinical studies, the administration of MSCs has been associated with reduced bacterial load and decreased levels of pro-inflammatory factors as well as the improved function of the different vital organs, including heart, kidney, liver, and lungs. The current study provides a brief review of sepsis and its pathophysiology, and then highlights recent findings in the therapeutic effects of MSCs and MSC-derived secretome in improving sepsis-induced organ dysfunction. Besides, eligible sepsis candidates for MSC-therapy and the latest clinical findings in these areas have been reviewed.

miRNA-146a Improves Immunomodulatory Effects of MSC-derived Exosomes in Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a severe inflammatory joint disorder, and several studies have taken note of the probability that microRNAs (miRNAs) play an important role in RA pathogenesis. MiR-146 and miR-155 arose as primary immune response regulators. Mesenchymal stem cells (MSCs) immunomodulatory function is primarily regulated by paracrine factors, such as exosomes. Exosomes, which serve as carriers of genetic information in cell-to-cell communication, transmit miRNAs between cells and have been studied as vehicles for the delivery of therapeutic molecules. AIMS: The current research aimed to investigate the therapeutic effect of miR-146a/miR-155 transduced mesenchymal stem cells (MSC)-derived exosomes on the immune response. METHODS: Here, exosomes were extracted from normal MSCs with over-expressed miR-146a/miR-155; Splenocytes were isolated from collagen-induced arthritis (CIA) and control mice. Expression levels miR-146a and miR-155 were then monitored. Flow cytometry was performed to assess the impact of the exosomes on regulatory T-cell (Treg) levels. Expression of some key autoimmune response genes and their protein products, including retinoic acid-related orphan receptor (ROR)-γt, tumor necrosis factor (TNF)-α, interleukin (IL)-17, -6, -10, and transforming growth factor (TGF)-ß in the Splenocytes was determined using both quantitative real-time PCR and ELISA. The results showed that miR-146a was mainly down-regulated in CIA mice. Treatment with MSC-derived exosomes and miR-146a/miR-155-transduced MSC-derived exosomes significantly altered the CIA mice Treg cell levels compared to in control mice. RESULTS: Ultimately, such modulation may promote the recovery of appropriate T-cell responses in inflammatory situations such as RA. CONCLUSION: miR-146a-transduced MSC-derived exosomes also increased forkhead box P3 (Fox- P3), TGFß and IL-10 gene expression in the CIA mice; miR-155 further increased the gene expressions of RORγt, IL-17, and IL-6 in these mice. Based on the findings here, Exosomes appears to promote the direct intracellular transfer of miRNAs between cells and to represent a possible therapeutic strategy for RA. The manipulation of MSC-derived exosomes with anti-inflammatory miRNA may increase Treg cell populations and anti-inflammatory cytokines.

HLA, Immune Response, and Susceptibility to COVID-19.

The severe acute respiratory syndrome caused by Coronavirus 2 (SARS-CoV-2) that appeared in December 2019 has precipitated the global pandemic Coronavirus Disease 2019 (COVID-19). However, in many parts of Africa fewer than expected cases of COVID-19, with lower rates of mortality, have been reported. Individual human leukocyte antigen (HLA) alleles can affect both the susceptibility and the severity of viral infections. In the case of COVID-19 such an analysis may contribute to identifying individuals at higher risk of the disease and the epidemiological level to understanding the differences between countries in the epidemic patterns. It is also recognized that first antigen exposure influences the consequence of subsequent exposure. We thus propose a theory incorporating HLA antigens, the "original antigenic sin (OAS)" effect, and presentation of viral peptides which could explain with differential susceptibility or resistance to SARS-CoV-2 infections.

Promotion of angiogenesis by M13 phage and RGD peptide in vitro and in vivo.

One of the most important goals of regenerative medicines is to generate alternative tissues with a developed vascular network. Endothelial cells are the most important cell type required in angiogenesis process, contributing to the blood vessels formation. The stimulation of endothelial cells to initiate angiogenesis requires appropriate extrinsic signals. The aim of this study was to evaluate the effects of M13 phage along with RGD peptide motif on in vitro and in vivo vascularization. The obtained results demonstrated the increased cellular proliferation, HUVECs migration, cells altered morphology, and cells attachment to M13 phage-RGD coated surface. In addition, the expression of Vascular Endothelial Growth Factor A (VEGF-A), VEGF Receptors 2 and 3, Matrix Metalloproteinase 9 (MMP9), and epithelial nitric oxide synthase (eNOS) transcripts were significantly upregulated due to the HUVECs culturing on M13 phage-RGD coated surface. Furthermore, VEGF protein secretion, nitric oxide, and reactive oxygen species (ROS) production were significantly increased in cells cultured on M13 phage-RGD coated surface.

Development of specific nanobodies (VHH) for CD19 immuno-targeting of human B-lymphocytes.

OBJECTIVES: CD19 is a transmembrane glycoprotein of immunoglobulin superfamily. In order to treat lymphoma, monoclonal antibodies (mAb) can target different antigens, including CD19, CD20 and CD22 on the surface of B-cells. Along with biotechnology progress, a new generation of antibodies is introduced, with the purpose of eliminating the defects of the previous generation. Among the most developed one are nanobodies (Nb). Nbs are a unique kind of camelid single domain antibody fragments with a broad range of medical applications. Unique physicochemical properties of Nbs have made them ideal candidates for therapeutic and diagnostic applications. MATERIALS AND METHODS: An immune gene library was created, and several CD19 specific Nbs were selected through antigen panning process, and their molecular properties as well as specificity, sensitivity, affinity and immunoreactivity against CD19 positive and negative cells were evaluated. RESULTS: The Nb library was prepared with 7.2 x107 members. We managed to isolate a panel of CD19-specific Nbs after the last round of selection with the affinity of isolated Nbs being estimated at the standard range of 15-35 nM. Sequence analysis of positive clones was indicative of the fact that 12 variable sequences were confirmed. Of all these 12 clones, 2 clones with the greatest level signal in ELISA underwent subsequent analysis. Our sequencing results indicated high sequence homology (approximately 90%) between the Nb and Homa variable immunoglobulin domains. CONCLUSION: Specific Nbs possess the potential to be used as novel therapeutic approaches in order to treat autoimmune diseases and B-cell lymphoma.

Altered microRNA Expression and Immunosuppressive Cytokine Production by Regulatory T Cells of Ulcerative Colitis Patients.

Regulatory T (Treg) cells are essential for maintenance of peripheral tolerance and prevention of autoimmune diseases in part by producing immunosuppressive cytokines. Recently, microRNAs (miRNAs) have also been involved in autoimmune disorders, not least for their crucial role in the regulation of Treg biology and function. We simultaneously investigated the concentration of IL-35, IL-10, TGF-ß, and sCD25 in supernatant of cell culture and the expression patterns of several miRNAs in CD4(+)CD25(+) CD127(-/low) FoxP3(+) Tregs of ulcerative colitis (UC) patients. Significantly lower levels of IL-10 and IL-35 were observed in Treg cultures of UC patients. miR-21, miR-146a, and miR-155 levels were downregulated and miR-31 level was upregulated in Tregs of patients. Our results suggest that microRNAs may serve as a novel regulator in function and homoeostasis of UC Treg cells, providing a key role for them in pathophysiology of UC.

Bioactivity and immunological evaluation of LPS from different serotypes of Helicobacter pylori.

BACKGROUND AND OBJECTIVES: Helicobacter pylori is the causative agent of peptic ulcer disease and a co-factor in development of gastric malignancies. LPS are among toxic substances produced by H. pylori exhibiting low endotoxic activity compared to typical bacterial LPS. The aim of this study was to investigate bioactivity of LPS produced by different serotypes of Helicobacter pylori compared to Escherichia coli and Brucella abortus LPS. MATERIALS AND METHODS: Bacterial LPS was extracted by the hot phenol-water method. Biological activities of LPS were determined via the limulus lysate assay, pyrogenic assay, and blood pressure and PBMC induction test in rabbits. RESULTS: Biological activity of O2 serotype LPS of H. pylori was less than the biological activity of other H. pylori serotypes. CONCLUSION: Our data supported the hypothesis that the unique bacterial LPS of the O2 serotype must be included in the formulation of a multivalent H. pylori vaccine.

Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System.

BACKGROUND: Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). METHODS: The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. RESULTS: Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. CONCLUSION: These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis.

Expression of human serum albumin--L7/L12 (Brucella abortus ribosomal protein) fusion protein in Saccharomyces cerevisiae.

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. It has already been expressed in several bacteria and has been used as DNA vaccine. In order to construct yeast expressing vector for the tHSA-L7/L12 fusion protein, the l7/l12 ribosomal gene was amplified by PCR. The expression plasmid pYtHSA-L7/L12 was constructed by inserting the L7/L12 gene into the pYHSA5 shuttle vector (containing inulinase signal sequence, HSA gene and Gal10 promoter). The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The secreted recombinant fusion protein was detected in supernatant by SDS-PAGE and confirmed by western blot analysis using anti-HSA and anti-L7/L12 antibodies. Fusion protein was purified by affinity chromatography and its amount was approximately 500 microg/liter.

Development and characterization of monoclonal antibodies against human epidermal growth factor receptor in Balb/c mice.

For production of monoclonal antibody (mAb) against human epidermal growth factor receptor (EGFR), first, five female Balb/c mice 6 to 8 weeks old were immunized against A431 tumoral cells that express more EGFR on its membrane in four periods and the most immune mouse was selected for fusion. The fusion of mouse's spleen immune cells with SP2/0 cells (myeloma cells) were fulfilled in presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells were screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Large scale of monoclonal antibodies was produced in vitro and ascetic fluid. In this investigation, 280 clones were obtained, 27 of which displayed absorbance more than 1. Of these, 3 clones represented absorbance about 1.7 and selected for limiting dilution. The yield of limiting dilution was 8 monoclones with absorbance about 2. These results indicate that such monoclonal antibodies against EGFR can be used in diagnosis and treatment of tumors with membranous EGFR.

Detection of lactoferrin in the neutrophils and plasma of the patients suffering from hepatitis C.

Lactoferrin (LF) has antimicrobial properties against bacteria, fungi and several viruses including herpes virus, HIV and hepatitis C virus. The aim of this study was to detect LF in PMNs and plasma of the patients suffering from hepatitis C and the healthy persons. The sonicated solutions of PMNs of two groups were evaluated by SDS-PAGE (10%), isoelectric focusing (30%) and dot blotting. The level of LF in plasma was measured by ELISA. The results confirmed the presence of LF in PMNs of the two groups. ELISA showed that the level of LF in plasma of patients was higher than normal persons. Based on these findings we conclude that not only the production of LF was not reduced in the patients but also its level was significantly increased compared with the normal persons(P less than 0.0001).

Production and characterization of monoclonal antibodies with specificity for human HLA-G isoforms.

Production of monoclonal antibodies to HLA-G, a nonpolymorphic antigen of non-classical HLA class I, is of basic and clinical importance. In the present study, monoclonal antibodies were prepared which recognize different membrane bound and soluble isoforms of HLA-G, following immunization of BALB/c mice with a synthetic peptide. Use of this peptide (23 residues), which is present in the alpha1 domain of HLA-G, was previously demonstrated to provide antibodies useful for recognition of HLA-G isoforms in human placenta. Antibody-producing hybridomas were screened by an indirect one-step ELISA method. A clone designated 5E6H7, secreted antibodies useful in immunostaining studies involving both surface HLA-G of placental tissues and soluble forms of this antigen in human sera. In addition, unreactivity of this antibody with human lymphocytes and sections of normal human skin was observed by immunofluorescence microscopy, thus demonstrating its specificity for HLA-G.

Immune responses against excreted/secreted antigens of Toxoplasma gondii tachyzoites in the murine model.

In the present study, excretory secretory antigens (ESA) of Toxoplasma gondii were evaluated in immunization of 8-10 week inbred female Balb/c mice. Tachyzoites of the parasite were cultured in cell-free incubation medium (RPMI-1640), and then supernatant of the medium was loaded on an ion-exchange chromatography column. Two fractions (ESA-F(1) and ESA-F(2)) were collected from the column. For immunization of the mice, 50 were allocated into 5 groups of 10. The first, second, third, and fourth groups were immunized, twice with total-ESA, ESA-F(1), ESA-F(2) or toxoplasma lysate antigen (TLA), respectively. The fifth group was selected as a negative control group (non-immunized). The virulent RH strain of Toxoplasma gondii was used to challenge. Delayed-type hypersensitivity responses (DTHs) were measured by intra-footpad injection measuring induration at timed intervals. Lymphocyte transformation tests (LTTs) were done on lymph node cells using [3H] thymidine incorporation as an indication of reactivity. Peritoneal macrophages from sensitized mice were stimulated and nitric oxide was measured by Griess method. The ESA-F(1) and ESA-F(2) fractions were separated on poly acrylamide gel electrophoresis (PAGE) and SDS-PAGE. ESA-F(1) had 4 bands on PAGE and 14 bands on SDS-PAGE. ESA-F(2) had one band on PAGE and two bands on SDS-PGE. Sensitized mice showed DTH and lymphocyte transformation responses to total-ESA, ESA-F(1), and ESA-F(2) and peritoneal macrophages produce nitric oxide following stimulation. In challenge experiments, all non-immunized mice died within 10 days, whereas immunized mice survived for longer time periods (P<0.05). The highest survival rate was observed in mice that immunized with ESA-F(2). We suggest that these antigens especially ESA-F(2) should be of value for the development of new strategies for immunization against toxoplasmosis.