Apoptotic cells for treatment of acute respiratory distress syndrome associated with COVID-19.

Background: Hyper-inflammatory immune response, a hallmark of severe COVID-19, is associated with increased mortality. Acute respiratory distress syndrome (ARDS) is a common manifestation. We undertook two phase I/II studies in five and then 16 subjects with severe/critical COVID-19 to assess the safety and preliminary efficacy of apoptotic cells (Allocetra™-OTS, Enlivex Therapeutics), a cellular immunomodulatory therapy that reprograms macrophages to reduce hyper-inflammatory response severity. Methods: Eligible patients presenting to the Emergency Room with severe COVID-19 and respiratory dysfunction received one intravenous administration of Allocetra™-OTS and were monitored for adverse events (AEs) for 28 days. The primary aim was to determine the safety profile of treatment; secondary aims were recovery from ARDS, intensive care unit (ICU) and hospital length-of-stay, and mortality. Immune modulator markers were measured to elucidate the mechanism of action of Allocetra™-OTS. Results: 21 patients with severe-critical COVID-19 of Gamma, Alpha and Delta variants, were treated with a single dose of apoptotic cells. 19/21 patients had mild-to-severe ARDS at presentation. Median age was 53 years, 16/21 were males, 16/21 were overweight/obese. No serious related adverse events (SAEs) were reported. All 21 study subjects survived to day 28 (end of study); 19/21 recovered completely. Comparable mortality rates at the hospital were 3.8%-8.9% for age- and gender-matched patients, and 39%-55% for critical patients. Recovering patients exhibited rapid ARDS resolution and parallel resolution of inflammation markers and elevated cytokines/chemokines. Conclusion: In patients with severe/critical COVID-19 associated with ARDS, Allocetra™-OTS was safe, well-tolerated, and showed promising results for resolution of respiratory failure and inflammation. Trial registration: https://clinicaltrials.gov/ct2/show/study/NCT04513470, https://clinicaltrials.gov/ct2/show/study/NCT04590053, Identifiers NCT04513470, NCT04590053.

Increased hepatic Akt phosphorylation alleviated glucose intolerance and improved liver function in leptin-deficient mice.

Aim of the study: Akt is involved in upregulating the insulin-signaling pathways essential for maintaining glucose metabolism. Glycosphingolipids are involved in the pathogenesis of glucose intolerance and associated target organ injury. On the other hand, oral administration of b-glucosylceramide (GC) has been shown to alleviate insulin resistance. The present study aimed to determine the effects of oral administration of insulin and GC, separately and in combination, on Akt expression and the subsequent effect on metabolic syndrome characteristics in leptin-deficient mice. Material and methods: Four groups of leptin-deficient ob/ob mice were orally administered for four weeks: vehicle, GC, short-acting insulin, and GC combined with insulin. Mice were followed for hepatic Akt expression and changes in tumor necrosis factor a (TNF-a) level, hyperlipidemia, and liver damage. Results: In mice that received insulin or GC, particularly those that received both, the liver phosphorylation of Akt was significantly increased compared to those that received only vehicle. Serum TNF-a levels decreased in insulin-treated mice. These effects were associated with alleviating glucose intolerance and hyperlipidemia, as manifested by a significant glucose tolerance test improvement and reductions in serum triglyceride and cholesterol levels. Significant liver damage alleviation was noted by liver enzyme reductions in all treated groups, along with liver steatosis in the insulin-treated mice. Conclusions: These data established the potential use of oral insulin administration with glycosphingolipids to alleviate glucose intolerance and associated liver damage and hyperlipidemia via increased Akt expression in the liver. The data support targeting Akt as a potent therapeutic target for metabolic syndrome.

Steroid-mediated liver steatosis is CD1d-dependent, while steroid-induced liver necrosis, inflammation, and metabolic changes are CD1d-independent.

INTRODUCTION: Glucocorticoids contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Natural killer T cells play a role in the pathogenesis of NAFLD and response to steroids. The present study aimed to determine the role of CD1d in steroid-mediated metabolic derangement and the steroid-protective effect of glycosphingolipids. METHODS: Ten groups of mice were studied. Steroids were orally administered to C57BL/6 mice to assess the therapeutic effect of ß-glucosylceramide (GC) on the development of steroid-mediated liver damage and metabolic derangements. The role of CD1d in the pathogenesis of steroid-induced liver damage and in mediating the hepatoprotective effect of GC was studied in CD1d-/- mice. RESULTS: A model of oral administration of steroids was established, resulting in insulin resistance, hyperinsulinemia, hypertriglyceridemia, liver steatosis, and hepatocellular injury. Steroid administration to CD1d-/- mice was associated with hyperglycemia and hypertriglyceridemia. However, CD1d-/- mice did not manifest marked steroid-induced steatosis. GC treatment alleviated steroid-associated metabolic derangements and liver injury independent of CD1d expression. CONCLUSION: A steroid-mediated model of NAFLD and metabolic derangements was established in which steroid-mediated steatosis was CD1d-dependent while steroid-induced liver necrosis, inflammation, and metabolic changes were CD1d-independent, which may support a dichotomy between steatosis and steatohepatitis in NAFLD.

Augmented antiviral T cell immunity by oral administration of IMM-124E in preclinical models and a phase I/IIa clinical trial: A method for the prevention and treatment of COVID-19.

Biological adjuvants that target the gut immune system are being developed for modulating the immune system. Hyperimmune bovine colostrum (HBC), produced by harvesting the bovine colostrum of dairy cows immunized to exogenous antigens, has been shown to modulate the immune responses and alleviate immune-mediated organ damages. The aim of the present study was to determine the ability of HBC to promote antiviral interferonγ (IFNγ) T cell responses. In a preclinical study, mice were orally administered with HBC for 5 days and tested for the number of T cell clones secreting IFNγ in response to viral antigens of the swine flu, New Caledonia influenza, and cytomegalovirus. In a phase I/IIa clinical trial, five healthy volunteers were treated for 5 days with HBC followed by testing the anti-coronavirus disease (COVID-19) immunity. In the preclinical study, oral administration of HBC augmented the number of T cell clones secreting IFNγ in response to viral antigens. In the clinical trial, oral administration of HBC to healthy males significantly increased the number of anti-COVID-19 spike protein IFNγ positive T cell clones. Oral administration of HBC provides a novel method for augmenting antiviral responses. Its high-safety profile makes it ideal for all disease stages and for pre-emptive therapy among medical personnel and other workers who are at a high risk of exposure to infections. The relatively low cost of HBC is expected to minimize care provider burdens, costs, and enable its global application.

Apoptotic Cells for Therapeutic Use in Cytokine Storm Associated With Sepsis- A Phase Ib Clinical Trial.

Background: Sepsis has no proven specific pharmacologic treatment and reported mortality ranges from 30%-45%. The primary aim of this phase IB study was to determine the safety profile of Allocetra™-OTS (early apoptotic cell) infusion in subjects presenting to the emergency room with sepsis. The secondary aims were to measure organ dysfunction, intensive care unit (ICU) and hospital stays, and mortality. Exploratory endpoints included measuring immune modulator agents to elucidate the mechanism of action. Methods: Ten patients presenting to the emergency room at the Hadassah Medical Center with sepsis were enrolled in this phase Ib clinical study. Enrolled patients were males and females aged 51-83 years, who had a Sequential Organ Failure Assessment (SOFA) score ≥2 above baseline and were septic due to presumed infection. Allocetra™-OTS was administered as a single dose (day +1) or in two doses of 140×106 cells/kg on (day +1 and +3), following initiation of standard-of-care (SOC) treatment for septic patients. Safety was evaluated by serious adverse events (SAEs) and adverse events (AEs). Organ dysfunction, ICU and hospital stays, and mortality, were compared to historical controls. Immune modulator agents were measured using Luminex® multiplex analysis. Results: All 10 patients had mild-to-moderate sepsis with SOFA scores ranging from 2-6 upon entering the study. No SAEs and no related AEs were reported. All 10 study subjects survived, while matched historical controls had a mortality rate of 27%. The study subjects exhibited rapid resolution of organ dysfunction and had significantly shorter ICU stays compared to matched historical controls (p<0.0001). All patients had both elevated pro- and anti-inflammatory cytokines, chemokines, and additional immune modulators that gradually decreased following treatment. Conclusion: Administration of apoptotic cells to patients with mild-to-moderate sepsis was safe and had a significant immuno-modulating effect, leading to early resolution of the cytokine storm. Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT03925857. (https://clinicaltrials.gov/ct2/show/study/NCT03925857).

Short-Term Cohousing of Sick with Healthy or Treated Mice Alleviates the Inflammatory Response and Liver Damage.

Cohousing of sick with healthy or treated animals is based on the concept of sharing an intestinal ecosystem and coprophagy, the consumption of feces, which includes sharing of the microbiome and of active drug metabolites secreted in the feces or urine. To develop a model for short-term cohousing, enabling the study of the effect of sharing an ecosystem on inflammatory states. To determine the impact of cohousing of sick and healthy mice on the immune-mediated disorders, mice injected with concanavalin A (ConA) were cohoused with healthy or sick mice or with steroid-treated or untreated mice. To determine the effect of cohousing on acetaminophen (APAP)-induced liver damage, APAP-injected mice were cohoused with N-acetyl-cysteine (NAC)-treated or untreated mice. In the ConA-induced immune-mediated hepatitis model, cohousing of sick with healthy mice was associated with the alleviation of liver damage in sick animals. Similarly, a significant decrease in serum ALT was noted in ConA-injected mice kept in the same cage as ConA-injected mice treated with steroids. A trend for reduction in liver enzymes in APAP-injected mice was observed upon cohousing with NAC-treated animals. Cohousing of sick mice with healthy or treated mice ameliorated the immune-mediated inflammatory state induced by ConA and APAP. These models for liver damage can serve as biological systems for determining the effects of alterations in the ecosystem on the immune system.

Correlations between components of the immune system.

Background: No evidence of the possibility of altering a constituent of the immune system without directly affecting one of its associated components has yet been shown. Methods: A schematic model was developed in which two triggers, fasting and splenectomy, were studied for their ability to affect the expression of cell membrane epitopes and the cytokine secretion of out-of-body autogeneic and syngeneic lymphocytes. Results: The effect of fasting and/or splenectomy on promoting correlations between immune systems was studied by determining the alterations in expressions of cell membrane epitopes and in cytokine secretion by out-of-body autogeneic and syngeneic lymphocytes. The effect of fasting as a trigger decreased expression of CD8 and CD25 and increased TNFα levels. The effect of splenectomy as a trigger was investigated in non-fasting mice by comparing splenectomized and non-splenectomized mice. An increase in the CD8 expression and in TNFα, IFNg, and IL10 secretion was noted. The effect of splenectomy as a trigger in fasting mice was determined by comparing splenectomized and non-splenectomized mice. Splenectomy significantly affected the expression of CD25 and CD4 CD25 and on secretion of TNFα, IFNg, and IL10. To determine the effect of keeping the cells in an out-of-body location on the expression of lymphocyte epitopes, tubes kept on top of the cages of the fasting mice were compared with tubes kept on top of empty cages. The results showed a significant change in the CD8 expression was noted. To determine the effect of keeping cells in an out-of-body location on cytokine secretion, tubes kept on cages were tested for cytokine levels significant decrease was noted in the secretion of TNFα and IFNg. Conclusions: The study showed that a mouse could affect cells at a distance and alter the expression of surface markers and cytokine secretion following two types of triggers: fasting and/or splenectomy. The data characterized a system for the induction of correlations between two's immune system components without a transfer of mediators. It suggests that an out-of-body correlation can be induced between two components of the immune system.

Glycosphingolipids Prevent APAP and HMG-CoA Reductase Inhibitors-mediated Liver Damage: A Novel Method for "Safer Drug" Formulation that Prevents Drug-induced Liver Injury.

Background and Aims: Acetaminophen (APAP) and HMG-CoA reductase inhibitors are common causes of drug-induced liver injury (DILI). This study aimed to determine the ability to reduce APAP- and statins-mediated liver injury by using formulations that combine glycosphingolipids and vitamin E. Methods: Mice were injected with APAP or with statins and treated before and after with ß-glucosylceramide (GC), with or without vitamin E. Mice were followed for changes in liver enzymes, liver histology, hepatic expression of JNK, STAT3 and caspase 3, as well as intrahepatic natural killer T cells (NKT) and the serum cytokine levels by flow cytometry. Results: Administration of GC before or after APAP alleviated the liver damage, as noted by a reduction of the liver enzymes, improvement in the liver histology and decreased hepatic caspase 3 expression. Beneficial effect was associated with a reduction of the intrahepatic NKT, JNK expression in the liver, and increased glutathione in the liver, and decreased TNF-α serum levels. Synergistic effect of co-administration of GC with vitamin E was observed. Similar protective effect of GC on statin-mediated liver damage was documented by a reduction in liver enzymes and improved liver histology, which was mediated by reduction of NKT, increased STAT3 expression in the liver, and reduced the TGF-ß and IL17 levels. Conclusions: ß-glycosphingolipids exert a hepatoprotective effect on APAP- and statins-mediated liver damage. Vitamin E exerted a synergistic effect to that of GC. The generation of "safer drug" formulations, which include an active molecule combined with a hepatoprotective adjuvant, may provide an answer to the real unmet need of DILI.

Alpha-1 Anti-trypsin Exerts a Hepatoprotective Effect on Immune-mediated Hepatitis and Acetaminophen-induced Liver Injury.

Background and Aims: The serine proteinase inhibitor alpha-1 anti-trypsin (AAT) protects the body against protease activity. Several functions of AAT beyond those attributed to its anti-protease activity have been described, among them immunomodulatory and anti-inflammatory properties. The present study aimed to determine the efficacy of AAT for the treatment of immune-mediated liver injury using the models of concanavalin A-induced immune-mediated hepatitis and acetaminophen -induced liver damage. Methods: AAT was administered to mice subjected to concanavalin A-induced immune-mediated hepatitis or 2 h after acetaminophen-induced liver damage. Mice were followed for changes in serum levels of liver enzymes, liver histology, and for interferon gamma serum levels. Results: Treatment with AAT alleviated concanavalin A-induced immune-mediated liver damage, as demonstrated by a reduction in the serum levels of liver enzymes and interferon gamma, and an improved lymphocyte infiltration into the liver on liver biopsies. Moreover, treatment with AAT was associated with alleviation of the acetaminophen-induced liver injury. Conclusions: AAT exerts an hepatoprotective effect on immune-mediated and drug-induced liver damage. The data support its potential use in patients with immune-associated liver disorders.

Oral Co-administration of Soy-derived Extracts with Alcohol or with Sugar-sweetened Beverages Exerts Liver and Sugar Protective Effects.

Background and Aims: Both alcoholic drinks and high sugar-containing soft drinks cause major health problems worldwide. Oral administration of OS and M1 soy-derived extracts has been shown to alleviate liver injury in animal models. The aim of the present study was to determine the liver- and sugar-protective effect of OS and M1 soy-derived extracts when added to alcohol and sugar-enriched drinks. Methods: Mice were treated with alcohol or high sugar-containing drinks, with and without administration of a combination of OS and M1 soy extracts. Mice were observed for the effects on liver injury, glucose metabolism, and the immune system. Results: Co-administration of the soy extracts OS and M1 significantly alleviated the liver injury induced by acute alcohol, as evidenced by decreased liver enzymes. These beneficial effects were associated with promotion of subsets of regulatory T lymphocytes and with a trend towards a pro-inflammatory to an anti-inflammatory cytokine shift. Co-administration of OS M1 soy extracts with sugar-sweetened beverages significantly alleviated the increases in serum sugar levels. Conclusions: OS and M1 extracts exert a synergistic hepato- and glucose-protective effect in models of alcohol-induced liver damage and soft drinks-associated increases in serum glucose. These extracts may provide a solution to the two pressing health problems.

A synergistic effect of Cremophor and beta glucosylceramide to exert liver and sugar protection.

Many commonly used drugs carry the potential to induce hepatotoxicity, and a large number of foods and beverages induce an increase in blood sugar levels. A change in lifestyle by omitting these compounds is not applicable in many circumstances. ß-Glucosylceramide (GC) is a naturally occurring glycosphingolipid that exerts an effect on the immune system. Cremophor EL (CrEL) is a synthetic, nonionic surfactant that is used as a vehicle for the administration of water-insoluble compounds. The aim of the present study was to determine the synergistic effect of oral administration of the combination of GC and CrEL (GCC) when added to potential toxic substrates or to sugar-enriched compounds. Four groups of mice, treated with PBS, GC, CrEL, or GCC were studied in the concanavalin A immune-mediated hepatitis model, in the acetaminophen (APAP) toxicity model, and as additives to two types of sugar-enriched soda drinks. Both GC and CrEL exerted hepatoprotective effects in the ConA hepatitis and APAP toxicity models. Moreover, in both models, GCC exerted a synergistic effect in ameliorating liver damage as manifested by a significant decrease in the ALT serum levels. When added to two types of sugar-enriched sodas, GCC exerted a synergistic effect in ameliorating the increase in blood sugar levels. GCC exerts synergistic hepatoprotective and glucose-protective effects in models of liver damage and increased serum glucose. GCC can provide liver and sugar protection when co-administered with hepatotoxic drugs or sugar-enriched drinks.

Extracts from Lentinula edodes (Shiitake) Edible Mushrooms Enriched with Vitamin D Exert an Anti-Inflammatory Hepatoprotective Effect.

Vitamin D has been known for its anti-inflammatory properties. Extracts derived from Lentinula edodes (Shiitake) edible mushroom exert an anti-inflammatory effect. These extracts contain high levels of ergosterol, which converts into ergocalciferol (vitamin D2) following exposure to ultraviolet light, followed by absorption and hydroxylation into the active form 25-hydroxyvitamin D [25(OH)D]. To determine the anti-inflammatory effect of overexpression of vitamin D in edible mushrooms, L. edodes mushrooms were exposed to ultraviolet-B light, freeze-dried, followed by measurement of vitamin D2 contents, in their dry weight. C57B1/6 mice were orally treated with vitamin D2-enriched or nonenriched mushroom extract prior and during concanavalin A-immune-mediated liver injury. Exposure to ultraviolet light increased vitamin D2 content in Shiitake edible mushrooms. Following feeding of vitamin D-enriched mushroom extracts to mice with immune-mediated hepatitis, a significant decrease in liver damage was noted. This was shown by a decrease in alanine aminotransferase and aspartate aminotransferase serum levels, a decrease in proportion of mice with severe liver injury, and by improvement in liver histology. These effects were associated with a decrease in serum interferon gamma levels. A synergistic effect was noted between the anti-inflammatory effect of the mushroom extracts and that of vitamin D. Oral administration of vitamin D-enriched L. edodes edible mushroom exerts a synergistic anti-inflammatory effect in the immune-mediated hepatitis. The data support its potential use as safe immunomodulatory adjuvant for the treatment of HCV and nonalcoholic steatohepatitis.

Altered distribution of regulatory lymphocytes by oral administration of soy-extracts exerts a hepatoprotective effect alleviating immune mediated liver injury, non-alcoholic steatohepatitis and insulin resistance.

AIM: To determine the immune-modulatory and the hepatoprotective effects of oral administration of two soy extracts in immune mediated liver injury and non-alcoholic steatohepatitis (NASH). METHODS: Two soy extracts, M1 and OS, were orally administered to mice with concanavalin A (ConA) immune-mediated hepatitis, to high-fat diet (HFD) mice and to methionine and choline reduced diet combined with HFD mice. Animals were followed for disease and immune biomarkers. RESULTS: Oral administration of OS and M1 had an additive effect in alleviating ConA hepatitis manifested by a decrease in alanine aminotransferase and aspartate aminotransferase serum levels. Oral administration of the OS and M1 soy derived fractions, ameliorated liver injury in the high fat diet model of NASH, manifested by a decrease in hepatic triglyceride levels, improvement in liver histology, decreased serum cholesterol and triglycerides and improved insulin resistance. In the methionine and choline reduced diet combined with the high fat diet model, we noted a decrease in hepatic triglycerides and improvement in blood glucose levels and liver histology. The effects were associated with reduced serum tumor necrosis factor alpha and alteration of regulatory T cell distribution. CONCLUSION: Oral administration of the combination of OS and M1 soy derived extracts exerted an adjuvant effect in the gut-immune system, altering the distribution of regulatory T cells, and alleviating immune mediated liver injury, hyperlipidemia and insulin resistance.

Oral Administration of OKT3 MAb to Patients with NASH, Promotes Regulatory T-cell Induction, and Alleviates Insulin Resistance: Results of a Phase IIa Blinded Placebo-Controlled Trial.

UNLABELLED: Oral administration of anti-CD3 antibodies induced regulatory T cells (Tregs) alleviating the insulin resistance and liver damage in animal models. OBJECTIVE: To determine the safety and biological effects of oral OKT3 monoclonal antibody (Balashov et al. Neurology 55:192-8, 2000) in patients with NASH. DESIGN: In this Phase-IIa trial, four groups of patients with biopsy-proven NASH (n = 9/group) received placebo (group A) or oral OKT3 (group B: 0.2; C: 1.0; D: 5.0 mg/day) for 30 days. Patients were followed for safety, liver enzymes, glucose, lipid profile, oral glucose tolerance test (OGTT), serum cytokines and Tregs. RESULTS: Oral OKT3 was well tolerated without treatment-related adverse events. OKT3 induced Tregs: with significant increases of CD4(+)LAP(+) (Latency associated peptide) and CD4(+)CD25(+)LAP(+) cells in Group D, and a significant increase in TGF-ß in Groups C and D. AST decreased significantly in group D and a trend in Groups B and C. Fasting plasma glucose decreased significantly in all treatment groups compared with placebo. OGTT decreased significantly in Group D. Correlations were observed between the changes in several immune-modulatory effects and clinical biomarkers. While serum anti-CD3 levels where undetectable increases in human anti-mouse antibody levels were observed in Groups C and D. CONCLUSION: Oral administration of anti-CD3 MAb to patients with NASH was safe and well tolerated. Positive biological effects were noted in several hepatic, metabolic and immunologic parameters. These findings provide the basis for future trials to investigate the effect of oral anti-CD3 MAb immunotherapy in patients with NASH.

Hepatoprotective effect of DT56a is associated with changes in natural killer T cells and regulatory T cells.

OBJECTIVE: To determine the metabolic and immunological effects of the oral administration of DT56a, an enzymatic isolate of soybeans. METHODS: DT56a was orally administered to mice in three animal models: leptin deficiency, high-fat diet (HFD) supplementation and immune-mediated hepatitis. Liver damage and immunological status were assessed. RESULTS: Oral administration of DT56a to leptin-deficient (ob/ob) and HFD mice led to a significant reduction in serum triglyceride (TG) and total cholesterol (TC) levels. DT56a-treated mice in both models exhibited a significant reduction in hepatic levels of TG and marked alleviation of glycemic control as indicated by significant decreases in fasting blood glucose levels and glucose tolerance tests. The levels of liver enzymes were reduced. These metabolic effects were associated with altered distributions of regulatory T (Tregs) and natural killer T (NKT) cells. DT56a suppressed the immune-mediated liver damage induced by concanavalin A indicated by decreased liver enzymes and serum interferon-γ levels and by improved histology and decreased hepatic apoptosis. Oral administration of DT56a also alleviated immune-mediated hepatitis and affected Tregs and NKT cells. CONCLUSIONS: Oral administration of DT56a promotes a hepatoprotective effect associated with an alteration in the distribution of Tregs and NKT cells.

Alleviation of insulin resistance and liver damage by oral administration of Imm124-E is mediated by increased Tregs and associated with increased serum GLP-1 and adiponectin: results of a phase I/II clinical trial in NASH.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is considered to be part of the nonalcoholic fatty liver disorders and its incidence is increasing. Imm124-E (Immuron Ltd, Melbourne, Australia), containing hyperimmune bovine colostrum, has been shown to exert an immunomodulatory effect and to alleviate target organ damage in animal models of NASH. The aim of our study was to determine the safety and efficacy of oral administration of Imm124-E to patients with insulin resistance and NASH. METHODS: In an open-label trial, ten patients with biopsy-proven NASH and insulin resistance were orally treated with Imm124-E for 30 days. RESULTS: Oral administration of Imm124-E was safe, and no side effects were noted. Alleviation of insulin resistance was reflected by significantly improved hemoglobin A(1c) (HbA(1c)) values in all ten treated patients. For between five and eight responders, the following effects were noted: a decrease in fasting glucose levels; improved oral glucose tolerance test (OGGT) and homeostatic model assessment insulin resistance (HOMA) scores; and alleviation in lipid profile. These effects were accompanied by increased serum levels of glucagon-like peptide 1 (GLP-1), adiponectin and T regulatory cells. CONCLUSION: Hyperimmune colostrum alleviates NASH.

Induction of graft-versus-leukemia (GVL) effect without graft-versus-host disease (GVHD) by pretransplant donor treatment with immunomodulators.

Pretransplant donor treatment with immunomodulators such as complete Freund's adjuvant (CFA) or oligodeoxynucleotide sequences expressing CpG motifs (CpG), was applied in sublethally irradiated host mice inoculated with murine models of mammary carcinoma (4T1) or B cell leukemia (BCL1). Spleen cells or IL-2 activated splenocytes (lymphokine activated killer [LAK]) derived from donor mice treated with CpG emulsified in incomplete Freund's adjuvant (IFA), (CpG + IFA) did not cause graft-versus-host disease (GVHD), but were not efficient enough to induce a significant graft-versus-tumor (GVT) response against 4T1 cells. In contrast, an efficient graft-versus-leukemia (GVL) effect was evident in BCL1-bearing mice inoculated with spleen cells from donors pretreated with CFA or CpG + IFA. Pretransplant donor treatment with CFA prolonged survival to a median of 62 days with 3 of 27 mice remaining GVHD- and leukemia-free for >200 days, compared to GVHD-related death of all mice inoculated with naïve cells (median 17 days), or leukemia-related death of all mice inoculated with leukemia cells (median of 27 days). Pretransplant donor treatment with CpG + IFA exerted a more efficient GVL effect with reduced GVHD resulting in 12 of 26 GVHD- and leukemia-free survivors for >200 days. Our results suggest that it may be possible to prevent GVHD while sparing an efficient GVL effect by using pretransplant donor treatment with immunomodulators prior to allogeneic stem cell transplantation and/or donor lymphocyte infusions in hematologic malignancies.

CpG-induced myeloid CD11b+Gr-1+ cells efficiently suppress T cell-mediated immunoreactivity and graft-versus-host disease in a murine model of allogeneic cell therapy.

Transplantation of mismatched allografts in irradiated recipients results in lethal graft- versus-host disease (GVHD). In our study, pretransplantation donor treatment with CpG, administered either alone or emulsified in incomplete Freund's adjuvant, efficiently prevented GVHD in sublethally irradiated recipients of haploidentical (H-2(b) into H-2(b/d)) and fully mismatched (H-2(b) into H-2(d)) allografts. CpG treatment of donor mice caused an accumulation of double-positive CD11bGr-1 cells in their blood and spleens, whereas treatment with CpG+IFA resulted in an even greater accumulation of these cells. Isolated CD11b(+) cells from the spleens of CpG+IFA-treated mice efficiently suppressed alloreactivity in vitro (> 92%), as determined by co-culturing these cells in mixed lymphocyte reactions. After CpG+IFA treatment, a T cell-depleted fraction enriched with CD11b(+)Gr-1(+) cells, acting as myeloid suppressor cells, was able to efficiently prevent GVHD induced by naïve T cells in the sublethally irradiated recipients: 20/21 mice remained GVHD-free survivors for more than 200 days. Splenocytes from CpG+IFA-treated mice displayed enhanced interleukin (IL)-6, IL-10, and interferon-gamma production, reduced T cell allogeneic and mitogenic responses, as well as failure of T cells to induce GVHD. In summary, CpG treatment led to impaired T cell function, enriched myeloid suppressor cells and regulatory cytokine production, which together appear to suppress alloreactivity and protect against the development of GVHD. We hypothesize that similar immunoregulatory effects could be applied experimentally in a clinical setting when inhibition of alloreactivity is required in recipients of stem cell allografts.