Insights into the Mechanism of Action of Highly Diluted Biologics.

The therapeutic use of Abs in cancer, autoimmunity, transplantation, and other fields is among the major biopharmaceutical advances of the 20th century. Broader use of Ab-based drugs is constrained because of their high production costs and frequent side effects. One promising approach to overcome these limitations is the use of highly diluted Abs, which are produced by gradual reduction of an Ab concentration to an extremely low level. This technology was used to create a group of drugs for the treatment of various diseases, depending on the specificity of the used Abs. Highly diluted Abs to IFN-γ (hd-anti-IFN-γ) have been demonstrated to be efficacious against influenza and other respiratory infections in a variety of preclinical and clinical studies. In the current study, we provide evidence for a possible mechanism of action of hd-anti-IFN-γ. Using high-resolution solution nuclear magnetic resonance spectroscopy, we show that the drug induced conformational changes in the IFN-γ molecule. Chemical shift changes occurred in the amino acids located primarily at the dimer interface and at the C-terminal region of IFN-γ. These molecular changes could be crucial for the function of the protein, as evidenced by an observed hd-anti-IFN-γ-induced increase in the specific binding of IFN-γ to its receptor in U937 cells, enhanced induced production of IFN-γ in human PBMC culture, and increased survival of influenza A-infected mice.

Translation of curative therapy concepts with T cell and cytokine antibody combinations for type 1 diabetes reversal in the IDDM rat.

Proinflammatory cytokines released from the pancreatic islet immune cell infiltrate in type 1 diabetes (T1D) cause insulinopenia as a result of severe beta cell loss due to apoptosis. Diabetes prevention strategies targeting different cytokines with antibodies in combination with a T cell antibody, anti-TCR, have been assessed for therapy success in the LEW.1AR1-iddm (IDDM) rat, an animal model of human T1D. Immediately after diabetes manifestation, antibody combination therapies were initiated over 5 days with anti-TNF-α (tumour necrosis factor), anti-IL-1ß (interleukin), or anti-IFN-γ (interferon) together with anti-TCR for the reversal of the diabetic metabolic state in the IDDM rat. Anti-TCR alone showed only a very limited therapy success with respect to a reduction of immune cell infiltration and beta cell mass regeneration. Anti-TCR combinations with anti-IL-1ß or anti-IFN-γ were also not able to abolish the increased beta cell apoptosis rate and the activated immune cell infiltrate leading to a permanent beta cell loss. In contrast, all anti-TCR combinations with anti-TNF-α provided sustained therapy success over 60 to 360 days. The triple combination of anti-TCR with anti-TNF-α plus anti-IL-1ß was most effective in regaining sustained normoglycaemia with an intact islet structure in a completely infiltration-free pancreas and with a normal beta cell mass. Besides the triple combination, the double antibody combination of anti-TCR with anti-TNF-α proved to be the most suited therapy for reversal of the T1D metabolic state due to effective beta cell regeneration in an infiltration free pancreas. KEY MESSAGES: Anti-TCR is a cornerstone in combination therapy for autoimmune diabetes reversal. The combination of anti-TCR with anti-TNF-α was most effective in reversing islet immune cell infiltration. Anti-TCR combined with anti-IL-1ß was not effective in this respect. The combination of anti-TCR with anti-TNF-α showed a sustained effect over 1 year.

Effect of interferon-ß on neuroinflammation, brain injury and neurological outcome after experimental subarachnoid hemorrhage.

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (SAH) has a poor outcome, particularly attributed to progressive injury after the initial incident. Several studies suggest a critical role for inflammation in lesion progression after SAH. Our goal was to test whether treatment with anti-inflammatory interferon-ß, which has shown promise as a therapeutic agent in experimental ischaemic stroke, can protect the brain after SAH. METHODS: SAH was induced in adult male Wistar rats by puncturing the intracranial bifurcation of the right internal carotid artery. Treatment effects of daily interferon-ß (n = 16) or vehicle (n = 14) injections were serially evaluated with multiparametric MRI and behavioral tests from day 0 to 7, in compliance with recent recommendations for pre-clinical drug testing. Outcome measures included neurological status, brain lesion volume, blood-brain barrier (BBB) leakage, and levels of inflammatory markers. RESULTS: In animals that survived up to 7 days post-SAH, we found no significant differences between vehicle- and interferon-ß-treated animals with respect to final neurological score (14.3 ± 1.0 vs. 13.0 ± 2.2), brain lesion size on T(2)-weighted MR images (59 ± 83 vs. 124 ± 99 mm(3)), BBB leakage (0.26 ± 0.05 vs. 0.22 ± 0.08 contrast-induced relative MR signal change), upregulation of brain RNA for cytokines, chemokines and cell adhesion molecules, and increased neutrophil activation. CONCLUSIONS: In contrast to previously published findings in experimental ischemic stroke models, interferon-ß has no clear efficacy to protect the brain after SAH. In line with recent highlighting of the significance of negative findings, our data currently do not recommend clinical testing of interferon-ß to prevent neurological damage in SAH patients.

Interferon-ß attenuates lung inflammation following experimental subarachnoid hemorrhage.

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (SAH) affects relatively young people and carries a poor prognosis with a case fatality rate of 35%. One of the major systemic complications associated with SAH is acute lung injury (ALI) which occurs in up to one-third of the patients and is associated with poor outcome. ALI in SAH may be predisposed by neurogenic pulmonary edema (NPE) and inflammatory mediators. The objective of this study was to assess the immunomodulatory effects of interferon-ß (IFN-ß) on inflammatory mediators in the lung after experimental SAH. METHODS: Male Wistar rats were subjected to the induction of SAH by means of the endovascular filament method. Sham-animals underwent sham-surgery. Rats received IFN-ß for four consecutive days starting at two hours after SAH induction. After seven days, lungs were analyzed for the expression of inflammatory markers. RESULTS: SAH induced the influx of neutrophils into the lung, and enhanced expression of the pulmonary adhesion molecules E-selectin, inter-cellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 compared to sham-animals. In addition, SAH increased the expression of the chemokines macrophage inflammatory protein (MIP)-1α, MIP-2, and cytokine-induced neutrophil chemoattractant (CINC)-1 in the lung. Finally, tumor necrosis factor-α (TNF-α) was significantly increased in lungs from SAH-animals compared to sham-animals. IFN-ß effectively abolished the SAH-induced expression of all pro-inflammatory mediators in the lung. CONCLUSIONS: IFN-ß strongly reduces lung inflammation after experimental SAH and may therefore be an effective drug to prevent SAH-mediated lung injury.

Granulocyte contamination dramatically inhibits spot formation in AIDS virus-specific ELISpot assays: analysis and strategies to ameliorate.

The interferon-gamma (IFNgamma) ELISpot assay has become the most critical tool for HIV vaccine evaluation. External factors affecting ELISpot results must be minimized for the data to be reliably used in vaccine research and development processes. In pre-clinical pigtail macaque studies analyzing HIV/SIV vaccine studies, we detected a strong correlation between levels of granulocytes contaminating PBMC preparations and reduction in the quality and quantity of spots in the IFNgamma ELISpot assay. Acute SHIV infection of macaques worsened granulocyte contamination of PBMC fractions and made the assay much less reliable in detecting SIV-specific T cell immunity compared to intracellular cytokine staining (ICS). This problem could be ameliorated by using an F(ab)(2) form of the MD-1 IFNgamma capture antibody, presumably reflecting that activation of granulocytes in the well by the Fc portion of the standard capture antibody disrupts spot formation. Improving the standard ELISpot assay by using an F(ab)(2) capture antibody should make it more reliable for use in critical vaccine development studies.

Interferon-beta blocks infiltration of inflammatory cells and reduces infarct volume after ischemic stroke in the rat.

The inflammatory response that exacerbates cerebral injury after ischemia is an attractive therapeutic target: it progresses over days and strongly contributes to worsening of the neurologic outcome. The authors show that, after transient ischemic injury to the rat brain, systemic application of interferon-beta (IFN-beta), a cytokine with antiinflammatory properties, attenuated the development of brain infarction. Serial magnetic resonance imaging (MRI) showed that IFN-beta treatment reduced lesion volume on diffusion-weighted MRI by 70% (P < 0.01) at 1 day after stroke. IFN-beta attenuated the leakage of contrast agent through the blood-brain barrier (P < 0.005), indicating a better-preserved blood-brain barrier integrity. Both control and IFN-beta-treated animals showed a similar degree of relative hyperperfusion of the lesioned hemisphere, indicating that neuroprotection by IFN-beta was not mediated by improved cerebral perfusion as assessed 24 hours after stroke onset. IFN-beta treatment resulted in an 85% reduction (P < 0.0001) in infarct volume 3 weeks later, as determined from T2-weighted MRI and confirmed by histology. This effect was achieved even when treatment was started 6 hours after stroke onset. Quantitative immunohistochemistry at 24 hours after stroke onset showed that IFN-beta almost completely prevented the infiltration of neutrophils and monocytes into the brain. Gelatinase zymography showed that this effect was associated with a decrease in matrix metalloproteinase-9 expression. In conclusion, treatment with the antiinflammatory cytokine IFN-beta affords significant neuroprotection against ischemia/reperfusion injury, and within a relatively long treatment window. Because IFN-beta has been approved for clinical use, it may be rapidly tested in a clinical trial for its efficacy against human stroke.

Interferon-beta prevents cytokine-induced neutrophil infiltration and attenuates blood-brain barrier disruption.

Inflammation can contribute to brain injury, such as that resulting from ischemia or trauma. The authors have previously shown that the cytokine interferon-beta (IFN-beta) affords protection against ischemic brain injury, which was associated with a diminished infiltration of neutrophils and a reduction in blood-brain barrier (BBB) disruption. The goal of the current study was to directly assess the effects of IFN-beta on neutrophil infiltration, with the use of an in vivo assay of neutrophil infiltration with relevance to ischemic brain injury. Intrastriatal injection of recombinant rat cytokine-induced neutrophil chemoattractant-1, a member of the interleukin-8 family (1 microg in 1 microl), triggered massive infiltration of neutrophils and extensive BBB disruption 6 hours later, as measured using immunofluorescence microscopy and magnetic resonance imaging in the rat, respectively. Depleting the animals of neutrophils before interleukin-8 injection prevented BBB disruption. Treatment with IFN-beta (5 x 106 U/kg) almost completely prevented neutrophil infiltration and attenuated BBB damage. Gelatinase zymography showed matrix metalloproteinase-9 expression in the ipsilateral striatum after interleukin-8 injection. Both neutrophil depletion and IFN-beta treatment downregulated matrix metalloproteinase-9. IFN-beta has already been approved for human use as a treatment for the chronic inflammatory disorder multiple sclerosis. The potential value of IFN-beta as a treatment that can attenuate acute brain inflammation is considered.

Transfected Plasmodium knowlesi produces bioactive host gamma interferon: a new perspective for modulating immune responses to malaria parasites.

Transgenic pathogenic microorganisms expressing host cytokines such as gamma interferon (IFN-gamma) have been shown to manipulate host-pathogen interaction, leading to immunomodulation and enhanced protection. Expression of host cytokines in malaria parasites offers the opportunity to investigate the potential of an immunomodulatory approach by generating immunopotentiated parasites. Using the primate malaria parasite Plasmodium knowlesi, we explored the conditions for expressing host cytokines in malaria parasites. P. knowlesi parasites transfected with DNA constructs for expressing rhesus monkey (Macaca mulatta) IFN-gamma under the control of the heterologous P. berghei apical membrane antigen 1 promoter, produced bioactive IFN-gamma in a developmentally regulated manner. IFN-gamma expression had no marked effect on in vitro parasite development. Bioactivity of the parasite-produced IFN-gamma was shown through inhibition of virus cytopathic effect and confirmed by using M. mulatta peripheral blood cells in vitro. These data indicate for the first time that it is feasible to generate malaria parasites expressing bioactive host immunomodulatory cytokines. Furthermore, cytokine-expressing malaria parasites offer the opportunity to analyze cytokine-mediated modulation of malaria during the blood and liver stages of the infection.

The frequency of interferon-gproducing cells reflects alloreactivity against minor histocompatibility antigens.

BACKGROUND: In human leukocyte antigen (HLA)-identical living-related renal transplant recipients, no donor-specific alloreactivity can be detected in regular tests (mixed lymphocyte culture, helper T-lymphocyte precursor frequencies, cytotoxic T-lymphocyte precursor frequencies) to identify patients responding to minor histocompatibility antigens (mHag). We questioned whether a more sensitive method like the Elispot-assay could be more appropriate. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMC) from 16 HLA-identical living-related kidney transplant patients 3 months (range, 2 weeks to 5 months) after transplantation were tested for the frequency of interferon (IFN)-gamma producing cells by the Elispot-assay. PBMC from the recipient were stimulated with irradiated donor PBMC and corrected for backward stimulation. Donor-specific IFN-gamma producing cells (pc) in the range of 5 to 115 per million PBMC (median, 30 per million PBMC) were found. To evaluate the frequency of spot forming cells in time, PBMC from six patients who received an HLA-identical renal transplant were stimulated with irradiated donor PBMC before, approximately 3 months after, and 12 months after transplantation. Four patients who received an HLA-mismatched living-related kidney served as positive control. In the HLA-identical group, frequencies in the range of 0 to 10 IFN-gamma pc per million PBMC were found before transplantation, 0 to 30 per million PBMC 3 months after transplantation, and 0 to 45 per million PBMC 12 months after transplantation. In the HLA-mismatched group, significantly higher numbers were found: 10 to 480 IFN-gamma pc per million PBMC before, 20 to 360 per million PBMC at 3 months, and 30 to 590 per million PBMC 12 months after transplantation. CONCLUSION: Under immunosuppressive therapy, IFN-gamma pc specific for donor mHag can be found after HLA-identical living-related renal transplantation.

The critical role of IL-12p40 in initiating, enhancing, and perpetuating pathogenic events in murine experimental autoimmune neuritis.

Interleukin 12 (IL-12) is a proinflammatory cytokine with important immunoregulatory activities and is critical in determining the differentiation and generation of Th1 cells. For the present study, we investigated the role of endogenous IL-12 in the pathogenesis of experimental autoimmune neuritis (EAN), which is a CD4+ T-cell mediated autoimmune inflammatory disease of the peripheral nervous system. EAN is used as an animal model for Guillain-Barré syndrome of humans. Here, EAN was established in IL-12 p40 deficient mutant (IL-12-/-) C57BL/6 mice by immunization with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. In these IL-12-/- mice the onset of clinical disease was delayed, and the incidence and severity of EAN were significantly reduced compared to that in wild-type mice.The former group's clinical manifestations were associated with less P0-peptide 180-199 induced secretion of interferon-gamma (IFN-gamma) by splenocytes in vitro and low production of anti-P0-peptide 180-199 IgG2b antibodies in serum. Fewer IFN-gamma and TNF-alpha producing cells, but more cells secreting IL-4, were found in sciatic nerve sections from IL-12-/- mice, consistent with impaired Th1 functions and response. However, the IL-12 deficiency appeared not to affect P0 peptide 180-199-specific T-cell proliferation. These results indicate that IL-12 has a major role in the initiation, enhancement and perpetuation of pathogenic events in EAN by promoting a Th1 cell-mediated immune response and suppressing the Th2 response. This information augments consideration of IL-12 as a therapeutic target in Guillain-Barré syndrome and other T-cell-mediated autoimmune diseases.

CD4 and CD8 T cells, but not B cells, are critical to the control of murine experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune disease of the peripheral nervous system that duplicates the clinical, pathological, and electrophysiological features of Guillain-Barré syndrome in humans. However, the molecular pathogenesis of EAN remains controversial. Therefore, for this study, we induced EAN with P0 protein peptide 180-199 in CD4(-/-), CD8(-/-), CD4(-)8(-), and B cell knockout (microMT) mice to further investigate the roles of these cells in EAN. Our results showed that the severity of clinical signs and histopathological manifestations of EAN and the T cell response to P0 peptide 180-199 in CD4(-/-) mice were significantly lower than those in their wild-type counterparts. CD8(-/-) mice also had a milder clinical course, less histopathological change, and a diminished T cell response to P0 peptide 180-199. However, more severe clinical and histopathological manifestations, a stronger T cell response to P0 peptide 180-199, and enhanced IFN-gamma production in the spleen were observed in the EAN of CD4(-)8(-) and microMT mice, but these were not obviously different from those of wild-type mice. Levels of IgG production were similar in sera from CD4(-/-), CD8(-/-), and CD4(-)8(-), and wild-type mice. These findings suggest that the induction and control of murine EAN are dependent on both CD4(+) and CD8(+) T cells and that B cells apparently do not perpetuate the related inflammatory demyelination.

Interferon-beta directly influences monocyte infiltration into the central nervous system.

Interferon-beta (IFN-beta) has beneficial effects on the clinical symptoms of multiple sclerosis (MS) patients, but its exact mechanism of action is yet unknown. We here suggest that IFN-beta directly modulates inflammatory events at the level of cerebral endothelium. IFN-beta treatment resulted in a marked reduction of perivascular infiltrates in acute experimental allergic encephalomyelitis (EAE), the rat model for MS, which was coupled to a major decrease in the expression of the adhesion molecules ICAM-1 and VCAM-1 on brain capillaries. In vitro, IFN-beta reduced the mRNA levels and protein expression of adhesion molecules of brain endothelial cell cultures and diminished monocyte transendothelial migration. Monocyte adhesion and subsequent migration was found to be predominantly regulated by VCAM-1. These data indicate that IFN-beta exerts direct antiinflammatory effects on brain endothelial cells thereby contributing to reduced lesion formation as observed in MS patients.

Stimulation of both humoral and cellular immune responses to HIV-1 gp120 by interleukin-12 in Rhesus macaques.

The adjuvant effect of recombinant Rhesus macaque interleukin-12 (RhIL-12) on the induction of cellular and humoral immune responses elicited by the HIV-1 subunit vaccine protein gp120 in Rhesus macaques was examined. RhIL-12 in conjunction with gp120 was given at day 0, 28 and 84 intramuscularly. Coadministration resulted in an approximate 10-fold increase in plasma anti-gp120 antibody levels as compared to levels generated in control monkeys receiving gp120 alone. Potentiation of the humoral arm of the immune response was evident by both ELISA and an antiviral bioassay. In addition, RhIL-12 was found to produce a significant increase in gp120-specific proliferative responses and in the frequency of antigen-specific IFN-gamma and IL-2 producing T cells after restimulation of PBMC with gp120 in vitro indicating that RhIL-12 potentiates cell-mediated immune responses as well. A critical finding was that during the course of the study, RhIL-12 did not induce a neutralizing antibody response to the administered cytokine. The doses of RhIL-12 were well tolerated and no detectable adverse side-effects on hematopoietic and hepatic parameters were noted. The data revealed that IL-12, when coadministered intramuscularly, acts as a potent adjuvant which is able to enhance not only cellular but also humoral immune responses to gp120 in non-human primates and may have to be considered in future HIV vaccine strategies.