Dual Checkpoint Aptamer Immunotherapy: Unveiling Tailored Cancer Treatment Targeting CTLA-4 and NKG2A.

Recent strides in immunotherapy have illuminated the crucial role of CTLA-4 and PD-1/PD-L1 pathways in contemporary oncology, presenting both promises and challenges in response rates and adverse effects. This study employs a computational biology tool (in silico approach) to craft aptamers capable of binding to dual receptors, namely, inhibitory CTLA4 and NKG2A, thereby unleashing both T and NK cells and enhancing CD8+ T and NK cell functions for tumor cell lysis. Computational analysis highlighted AYA22T-R2-13 with HADDOCK scores of -78.2 ± 10.2 (with CTLA4), -60.0 ± 4.2 (with NKG2A), and -77.5 ± 5.6 (with CD94/NKG2A). Confirmation of aptamer binding to targeted proteins was attained via ELISA and flow cytometry methods. In vitro biological functionality was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. Direct and competitive assays using ELISA and flow cytometry demonstrated the selective binding of AYA22T-R2-13 to CTLA4 and NKG2A proteins, as well as to the cell surface receptors of IL-2-stimulated T cells and NK cells. This binding was inhibited in the presence of competition from CTLA4 or NKG2A proteins. Remarkably, the blockade of CTLA4 or NKG2A by AYA22T-R2-13 augmented human CD8 T cell- and NK cell-mediated tumor cell lysis in vitro. Our findings highlight the precise binding specificity of AYA22T-R2-13 for CTLA4-B7-1/B7-2 (CD80/CD86) or CD94/NKG2A-HLA-E interactions, positioning it as a valuable tool for immune checkpoint blockade aptamer research in murine tumor models. These in vitro studies establish a promising foundation for further enhancing binding capacity and establishing efficacy and safety in animal models. Consequently, our results underscore the potential of AYA22T-R2-13 in cancer immunotherapy, offering high specificity, low toxicity, and the potential for cost-effective production.

T helper (Th) cell profiles and cytokines/chemokines in characterization, treatment, and monitoring of autoimmune diseases.

Autoimmune diseases (AD) consist of a spectrum of disease entities whose etiologies are very complex and still not well understood. Every individual has the potential for developing AD under appropriate conditions because the body contains lymphocytes that are potentially reactive with self-antigens. The aims of this study are to (1) explore the flow cytometry method to identify the frequency of various circulating CD4+ T helper (Th) cell-subsets, including Th1, Th2, Th9, Th17, Th17.1, and Th22; (2) In parallel, to examine multiplex ELISA method for pathogenic inflammatory cytokines/chemokines, and (3) To assess the correlation of expression of T cell-subsets with serum cytokines/chemokines and understand its clinical importance with available AD treatments. We analyzed Th17, Th17.1, Th22, Th2, Th1, and Th9 Th cell populations and compared the concentrations of 67 cytokines/chemokines in healthy as well as AD-diagnosed patients. We observed that patients with autoimmune markers had significantly elevated percentages of naïve (Th17, Th22, and Th9) as well as memory (Th17 and Th22) Th cell-subsets, along with increased concentrations of cytokines/chemokines (Eotaxin, TNFß, and FABP4). The percentage of Th cell-subsets correlated positively or negatively with the production of cytokines/chemokines of patients diagnosed with AD. Our study demonstrates that the naïve and memory Th cell-subsets with positive correlations to cytokines/chemokines show new diagnostic markers to predict the patients' outcome, while the negative correlation of cytokines/chemokines shows the response to autoimmune therapies. Our findings of Th cell-subsets by flow cytometry and cytokines/chemokines by multiplex ELISA suggest that CCR6+ Th cell-subsets (Th17, Th17.1, Th22, and Th9) contribute to our understanding of the pathogenesis of AD and identify the new onset of AD from the autoimmune spectrum. Our findings highlight the importance of CCR6+ as a possible marker in the characterization, treatment, and monitoring of AD.

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote.

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.

Highly efficacious and safe neutralizing DNA aptamer of SARS-CoV-2 as an emerging therapy for COVID-19 disease.

BACKGROUND: The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L. METHODS: In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice. RESULTS: We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies. CONCLUSIONS: Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.

Noninvasive nasopharyngeal proteomics of COVID-19 patient identify abnormalities related to complement and coagulation cascade and mucosal immune system.

Serum or plasma have been the primary focus of proteomics studies for COVID-19 to identity biomarkers and potential drug targets. The nasal mucosal environment which consists of lipids, mucosal immune cells, and nasal proteome, has been largely neglected but later revealed to have critical role combating SARS-CoV-2 infection. We present a bottom-up proteomics investigation of the host response to SARS-CoV-2 infection in the nasopharyngeal environment, featuring a noninvasive approach using proteins in nasopharyngeal swabs collected from groups of 76 SARS-CoV-2 positive and 76 negative patients. Results showed that 31 significantly down-regulated and 6 up-regulated proteins were identified (p < 0.05, log2 FC > 1.3) in SARS-CoV-2 positive patient samples as compared to the negatives; these proteins carry potential value as markers for the early detection of COVID-19, disease monitoring, as well as be drug targets. The down-regulation of coagulation factor 5 indicates a thrombotic abnormality in COVID-19 patients and the decreased IgG4 suggests an abnormal immune response at the point of entry in human nasopharyngeal environment, which is in consistent with KEGG and GO pathway analysis. Our study also demonstrated that mass spectrometry proteomics analysis of nasopharyngeal swabs can be used as a powerful early approach to evaluate host response to SARS-CoV-2 viral infection.

A Single-Domain TCR-like Antibody Selective for the Qa-1b/Qdm Peptide Complex Enhances Tumoricidal Activity of NK Cells via Blocking the NKG2A Immune Checkpoint.

The NKG2A/HLA-E axis is an immune checkpoint that suppresses immune effector activity in the tumor microenvironment. In mice, the ligand for the NKG2A/CD94 inhibitory receptor is the nonclassical MHC molecule Qa-1b, the HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. To broaden our understanding of Qa-1b/Qdm peptide complex biology and its tumor protective role, we identified a TCR-like Ab from a single domain VHH library using yeast surface display. The TCR-like Ab (EXX-1) binds only to the Qa-1b/Qdm peptide complex and not to Qa-1b alone or Qa-1b loaded with control peptides. Conversely, currently available Abs to Qa-1b bind independent of peptide loaded. Flow cytometric results revealed that EXX-1 selectively bound to Qa-1b/Qdm-positive B16F10, RMA, and TC-1 mouse tumor cells but only after pretreatment with IFN-γ; no binding was observed following genetic knockdown of Qa-1b or Qdm peptide. Furthermore, EXX-1 Ab blockade promoted NK cell-mediated tumor cell lysis in vitro. Our findings show that EXX-1 has exquisite binding specificity for the Qa-1b/Qdm peptide complex, making it a valuable research tool for further investigation of the Qa-1b/Qdm peptide complex expression and regulation in healthy and diseased cells and for evaluation as an immune checkpoint blocking Ab in syngeneic mouse tumor models.

Central Roles of OX40L-OX40 Interaction in the Induction and Progression of Human T Cell-Driven Acute Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is one of the major obstacles for the success of allogeneic hematopoietic stem cell transplantation. Here, we report that the interaction between OX40L and OX40 is of critical importance for both induction and progression of acute GVHD (aGVHD) driven by human T cells. Anti-human OX40L monoclonal antibody (hOX40L) treatment could thus effectively reduce the disease severity in a xenogeneic-aGVHD (x-aGVHD) model in both preventative and therapeutic modes. Mechanistically, blocking OX40L-OX40 interaction with an anti-hOX40L antibody reduces infiltration of human T cells in target organs, including liver, gut, lung, and skin. It also decreases IL-21- and TNF-producing T cell responses, while promoting regulatory T cell (Treg) responses without compromising the cytolytic activity of CD8+ T cells. Single blockade of hOX40L was thus more effective than dual blockade of IL-21 and TNF in reducing the severity of aGVHD as well as mortality. Data from this study indicate that OX40L-OX40 interactions play a central role in the pathogenesis of aGVHD induced by human T cells. Therapeutic strategies that can efficiently interrupt OX40L-OX40 interaction in patients might have potential to provide patients with an improved clinical benefit.

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

PURPOSE: Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. METHODS: Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 µg/mL), PAR1 agonists (thrombin, 10 µM; TRAP-6, 10 µM), and PAR2 agonists (SLIGRL-NH2, 100 µM; AC 55541, 10 µM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 µM) and PAR2 (FSLLRY-NH2, 100 µM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. RESULTS: Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist (P < 0.05). Protease-activated receptor 1 antagonist did not alter aPA-stimulated IL-8 mRNA expression and protein production in HCE cells. Flow cytometry and immunocytochemistry showed that aPA and SLIGRL-NH2 (PAR2 agonist) upregulated PAR2 surface protein as compared to that in unstimulated HCE cells. Thrombin, but not aPA, stimulated PAR1 surface protein in HCE cells. CONCLUSIONS: Acanthamoeba plasminogen activator specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway, and PAR2 antagonists may be used as a therapeutic target in AK.

Pathogenic strains of Acanthamoeba are recognized by TLR4 and initiated inflammatory responses in the cornea.

Free-living amoebae of the Acanthamoeba species are the causative agent of Acanthamoeba keratitis (AK), a sight-threatening corneal infection that causes severe pain and a characteristic ring-shaped corneal infiltrate. Innate immune responses play an important role in resistance against AK. The aim of this study is to determine if Toll-like receptors (TLRs) on corneal epithelial cells are activated by Acanthamoeba, leading to initiation of inflammatory responses in the cornea. Human corneal epithelial (HCE) cells constitutively expressed TLR1, TLR2, TLR3, TLR4, and TLR9 mRNA, and A. castellanii upregulated TLR4 transcription. Expression of TLR1, TLR2, TLR3, and TLR9 was unchanged when HCE cells were exposed to A. castellanii. IL-8 mRNA expression was upregulated in HCE cells exposed to A. castellanii. A. castellanii and lipopolysaccharide (LPS) induced significant IL-8 production by HCE cells as measured by ELISA. The percentage of total cells positive for TLR4 was higher in A. castellanii stimulated HCE cells compared to unstimulated HCE cells. A. castellanii induced upregulation of IL-8 in TLR4 expressing human embryonic kidney (HEK)-293 cells, but not TLR3 expressing HEK-293 cells. TLR4 neutralizing antibody inhibited A. castellanii-induced IL-8 by HCE and HEK-293 cells. Clinical strains but not soil strains of Acanthamoeba activated TLR4 expression in Chinese hamster corneas in vivo and in vitro. Clinical isolates but not soil isolates of Acanthamoeba induced significant (P< 0.05) CXCL2 production in Chinese hamster corneas 3 and 7 days after infection, which coincided with increased inflammatory cells in the corneas. Results suggest that pathogenic species of Acanthamoeba activate TLR4 and induce production of CXCL2 in the Chinese hamster model of AK. TLR4 may be a potential target in the development of novel treatment strategies in Acanthamoeba and other microbial infections that activate TLR4 in corneal cells.

Significance of arachidonic acid in ocular infections and inflammation.

Innate immune responses in the cornea mainly play an important role to mobilize multiple interrelated pathways of corneal lipid, which involve in inflammatory corneal diseases. Signaling lipid mediators derived from arachidonic acid (AA) control cell proliferation, apoptosis, metabolism, and migration, are known as eicosanoids, phosphoinositides, sphingolipids, and fatty acids. Emerging evidences have highlighted the implication of lipid mediators in both injury and repair mechanisms in the cornea. Recently, the role of AA and its metabolites to induce proinflammatory mediators and inflammatory cell infiltration in the pathogen-infected cornea and to cause severe keratitis have been revealed. In this review, we focus on the novel roles of AA downstream signaling in the corneal inflammatory diseases and also the biological relevance of AA signaling in the therapeutic strategies for targeting sight-threatening diseases.

Role of protease-activated receptors 2 (PAR2) in ocular infections and inflammation.

Protease-activated receptors (PARs) belong to a unique family of G protein-coupled receptors (GPCRs) that are cleaved at an activation site within the N-terminal exodomain by a variety of proteinases, essentially of the serine (Ser) proteinase family. After cleavage, the new N-terminal sequence functions as a tethered ligand, which binds intramolecularly to activate the receptor and initiate signaling. Cell signals induced through the activation of PARs appear to play a significant role in innate and adoptive immune responses of the cornea, which is constantly exposed to proteinases under physiological or pathophysiological conditions. Activation of PARs interferes with all aspects of the corneal physiology such as barrier function, transports, innate and adoptive immune responses, and functions of corneal nerves. It is not known whether the proteinase released from the microorganism can activate PARs and triggers the inflammatory responses. The role of PAR2 expressed by the corneal epithelial cells and activation by serine protease released from microorganism is discussed here. Recent evidences suggest that activation of PAR2, by the serine proteinases, play an important role in innate and inflammatory responses of the corneal infection.

Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis.

The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (P < 0.05). Additionally, cPLA2α inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P < 0.05). Subconjunctival injection of purified MIP-133 in Chinese hamster eyes induced cytopathic effects resulting in corneal ulceration. Animals infected with A. castellanii-laden contact lenses and treated with AACOCF3 and CAY10650, showed significantly less severe keratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis.

Biochemical and histopathological evaluation of histamine receptors (H1R, H2R, H3R and H4R)-agonist in rabbits.

The present study was designed to investigate the biochemical and histopathological changes in the livers of rabbits treated with histamine and histamine receptors (H1R-H4R)-agonist. The cohort comprised of six groups containing five rabbits each. Control group received sterile distilled water (1 mL/kg × b.i.d.) and treated groups received subcutaneous histamine (100 µg/kg × b.i.d.) and H1R-H4R-agonist (histamine trifluoro-methyl toluidide, amthamine, R-[-]-α-methylhistamine, clobenpropit, respectively) each in a dose of 10 µg/kg × b.i.d. (12 h [8 am and 8 pm]) for 30 days. Hepatotoxicity due to these agonists was analyzed using biochemical and histopathological methods. Histamine and H1R-H3R-agonist were found to be hepatotoxic as shown by statistically significant (p < 0.05) elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), most marked in the H3R-agonist group. However, their levels in H4R-agonist group remained very similar to the control group. The entire drug treated groups as compared to control showed significant elevated levels of alkaline phosphatase (ALP). Histopathological examination revealed obvious changes in histamine, H2R- and H3R-agonist groups in terms of alteration of hepatic microstructure, congestion, focal necrosis and increased incidence of multinucleate hepatocytes while H1R and H4R groups showed minimal changes. From the findings of the present study it may be concluded that on repeated administration, histamine and HR-agonists-induced hepatotoxicity which is most pronounced with H3R-agonist though not severe enough to jeopardize the vital functions of liver and warrants further long-term studies.

Acanthamoeba-cytopathic protein induces apoptosis and proinflammatory cytokines in human corneal epithelial cells by cPLA2α activation.

PURPOSE: We have shown that Acanthamoeba interacts with a mannosylated protein on corneal epithelial cells and stimulates trophozoites to secrete a mannose-induced 133 kDa protease (MIP-133), which facilitates corneal invasion and induces apoptosis. The mechanism of MIP-133-induced apoptosis is unknown. The aim of this study was to determine if MIP-133 induces apoptosis and proinflammatory cytokines/chemokines in human corneal epithelial (HCE) cells via the cytosolic phospholipase A(2α) (cPLA(2α)) pathway. METHODS: HCE cells were incubated with or without MIP-133 at doses of 7.5, 15, and 50 µg/mL for 6, 12, and 24 hours. The effects of cPLA(2α) inhibitors on cPLA(2α), arachidonic acid (AA) release, and apoptosis were tested in vitro. Inhibition of cPLA(2α) involved preincubating HCE cells for 1 hour with cPLA(2α) inhibitors (10 µM methyl-arachidonyl fluorophosphonate [MAFP] or 20 µM arachidonyl trifluoromethyl ketone [AACOCF3]) with or without MIP-133 for 24 hours. Expression of cPLA(2α) mRNA and enzyme was examined by RT-PCR and cPLA(2) activity assays, respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 and DNA fragmentation assays. Expression of IL-8, IL-6, IL-1ß, and IFN-γ was examined by RT-PCR and ELISA. RESULTS: MIP-133 induced significant cPLA(2α) (approximately two to four times) and AA release (approximately six times) from corneal cells while cPLA(2α) inhibitors significantly reduced cPLA(2α) (approximately two to four times) and AA release (approximately three times) (P < 0.05). cPLA(2α) inhibitors significantly inhibited MIP-133-induced DNA fragmentation approximately 7 to 12 times in HCE cells (P < 0.05). MIP-133 specifically activates cPLA(2α) enzyme activity in HCE cells, which is blocked by preincubation with anti-MIP-133 antibody. In addition, MIP-133 induced significant IL-8, IL-6, IL-1ß, and IFN-γ production, approximately two to three times (P < 0.05). CONCLUSIONS: MIP-133 interacts with phospholipids on plasma membrane of HCE cells and activates cPLA(2α). cPLA(2α) is involved in apoptosis, AA release, and activation of proinflammatory cytokines/chemokines from HCE cells. cPLA(2α) inhibitors may be a therapeutic target in Acanthamoeba keratitis.

Dose-dependent effect of histamine on liver function markers in immunized rabbits.

OBJECTIVE: The present study was designed to delineate the hepatotoxicological roles of histamine dose-dependently in immunized rabbits. METHODS: The cohort comprised of three groups (II, III and IV), containing 18 rabbits each, and received subcutaneous histamine 50 µg/kg, 100 µg/kg and 200 µg/kg, respectively for 10 days (b.i.d., starting from 3 days prior to immunization until 7 days after immunization). Group I (control, n=18) received subcutaneous sterile distilled water for 10 days. They were subsequently immunized at day 3 with intravenous injection of SRBC (1×10(9) cells/ml). Blood samples were collected on pre-immunization (pre-I) day 0, as well as on days 7-, 14-, 21-, 28- and 58-post-immunization (post-I). Biochemical parameters aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin [total bilirubin (TB), direct bilirubin (DB) and indirect bilirubin (IB)] were determined. RESULTS: Groups II and IV revealed a significant decrease (on day 0-pre-I) and a significant increase (on days 7-, 14-, 21-, 28- and 58-post-I) in ALT and AST levels, when compared with the corresponding values of groups I and III while group II showed a significant increase in ALT and AST levels as compared to group IV. ALP levels in groups II, III and IV showed a significant enhancement when compared with group I. Moreover, results of TB, DB and IB demonstrated increased levels in group III when compared with groups I, II and IV. The results were found statistically significant (p<0.05). CONCLUSION: Short-term treatment of histamine produces dose-dependent differential patterns of hepatic dysfunctions suggestive mild liver degeneration warranting further long-term studies.

Immunological, biochemical and histopathological evaluation of histamine receptors (H1R, H2R, H3R and H4R)-antagonist in rabbit experimental model: a short term study.

The present study was designed to delineate the immuno- and hepatotoxicological roles of HRs-antagonists in vivo which is elementary in existing literature. The cohort comprised of two experimental studies. Experimental study 1 was designed for immunological investigations and consisted of seven groups and immunized with intravenous injection of SRBC at day 3 containing six rabbits each. Experimental study 2 was designed to assess the functional status of liver and comprised of seven groups containing five rabbits each. In both experimental studies group-I received sterile distilled water intramuscularly, and group II-VI received subcutaneous histamine, pheniramine (H1R-antagonist), ranitidine (H2R-antagonist), iodophenpropit (H3R-antagonist) and JNJ7777120 (H4R-antagonist), respectively while group-VII received DMSO intramuscularly. ELISA was used to assess the immunological investigations. The SRBC-specific immunoglobulins (Igs), IgM and IgG were significantly increased (p<0.05). Hepatotoxicity due to same histamine and HRs-antagonists were demonstrated by biochemical and histopathological methods. Rabbits in group II-VI had significantly (p<0.05) elevated levels of serum enzymes (ALT, AST, ALP) and bilirubin. Histopathological examination showed maintained hepatic lobular architecture in histamine and DMSO-treated groups a kin to control. Notable findings in other groups included increased binuclearity in H1R, trinuclearity in H2R, oxyphilic clusters of hepatocytes in H3R and moderate centrilobular necrosis in H3R and H4R-antagonist-treated groups without obvious inflammatory cell infiltration and Kupffer cell prominence. It is concluded that HRs-antagonist play immune suppressive role through H1R, H2R and H4R while immune enhancing role through H3R. In addition, HRs-antagonists appear moderately hepatotoxic in terms of altered serum enzyme levels and non-inflammatory hepatocellular damage.

The Influence of histamine H1-receptor on liver functions in immunized rabbits.

This study was designed to investigate the functional roles of histamine and histamine H1-receptor agonist and antagonist in the development of liver function impairment in immunized rabbits. The study comprised of six groups containing 18 rabbits each. Group III-VI received histamine (100 µg/kg, s.c.), H1R-agonist (HTMT, 10 µg/kg, s.c.), H1R-antagonist (pheniramine, 10 mg/kg, i.m.), and H1R-antagonist (pheniramine, 10 mg/kg, i.m.) plus histamine (100 µg/kg, s.c.), respectively, b.i.d. for 10 days. Group I (negative control) and group II (positive control) received sterile distilled water intramuscularly b.i.d. for 10 days. Groups II-VI were immunized on day 3 with intravenous injection of SRBC (1 × 10(9) cells/ml). Blood samples were collected on pre-immunization day 0, as well as on days 7-, 14-, 21-, 28-, and 58-post-immunization. Biochemical parameters AST, ALT, alkaline phosphatase and bilirubin [total bilirubin (TB), direct bilirubin (DB), and indirect bilirubin (IB)] were determined. On each experimental day, the mean values of serum enzymes and bilirubin in group I and group II showed no significant changes while in group III, IV, V, and VI, these enzymes and bilirubin levels showed significant changes (p < 0.05), when compared with their experimental values within the group. The levels of serum enzymes and bilirubin showed significant difference (p < 0.05) in group III, IV, V, and VI on each experimental day, when compared with the corresponding values of each other, and also compared with the corresponding values of group I and II. Histamine, HTMT, pheniramine, and combination of histamine + pheniramine cause hepatic function impairment in terms of altered serum enzymes and bilirubin levels. The present findings suggest that HTMT causes moderate liver function impairment while others show mild impairment.

The oxidative by-product, hydroxyl radical, damaged immunoglobulin-G in patients with non-insulin dependent diabetes mellitus.

BACKGROUND AND OBJECTIVE: Free radical-mediated oxidative damage and consequent protein modifications are important mediators of cell toxicity and disease pathogenesis. Earlier investigations from our laboratory revealed an increased oxidative damage in diabetes patients and hydroxyl radicals (OH) caused an extensive damage to immunoglobulin G (IgG) and oxidatively modified IgG was found to be highly immunogenic in rabbits. Here, for the first time, we evaluated the role of hydroxyl radical-damaged IgG (OH-IgG) in non insulin dependent (Type 2) diabetes patients. METHODS: IgG was isolated from normal human serum by affinity chromatography and modified by hydroxyl radicals, generated by UV irradiation of hydrogen peroxide. Type 2 diabetes patients (n = 38) were accessed by direct binding and competitive inhibition ELISAs and the results were compared to healthy age and sex matched human subjects (n = 22). RESULTS: A high degree of specific binding by 31.6% of diabetes sera antibodies towards OH-IgG was observed, in comparison to its native analog (p < 0.01). Antibodies from normal human subjects showed negligible binding with either of antigens (p > 0.05). CONCLUSION: Our results suggest that hydroxyl radicals' modification of IgG causes structural perturbations, resulting in the generations of neo-epitopes, and making it a potential immunogen. The IgG modified with hydroxyl radicals may be one of the factors for the induction of circulating type 2 diabetes autoantibodies. The OH-IgG may proves one of the best markers to determine the oxidative stress in type 2 diabetes patients (Tab. 1, Fig. 4, Ref. 44).

Modulation of in vivo immunoglobulin production by endogenous histamine and H1R and H2R agonists and antagonists.

The present study was designed to delineate the immunomodulatory role of histamine receptors (H1R and H2R) and their antibody generation in a rabbit model. Six groups containing 18 rabbits each received either vehicle (sterile distilled water, 1 ml/kg x b.i.d), histamine (100 µg/kg x b.i.d.), H1R agonist (HTMT, 10 µg/kg x b.i.d.), H2R agonist (amthamine, 10 µg/kg x b.i.d.), H1R antagonist (pheniramine, 10 mg/kg x b.i.d.) or H2R antagonist (ranitidine, 10 mg/kg x b.i.d.). All animals were subsequently immunized with an intravenous injection of sheep red blood cells (SRBC). Estimations of total serum immunoglobulins (Igs), immunoglobulin M (IgM) and immunoglobulin G (IgG) were performed by ELISA and hemagglutination assay (HA) at days 0 (pre-immunization), 7, 14, 21, 28 and 58 (post-immunization). Both the ELISA and the HA showed similar production of Igs, IgM and IgG but the results were found comparatively more significant by ELISA as opposed to HA. Results showed that histamine could influence a detectable antibody response to SRBC early (i.e., at day 7), which lasted until day 58. Immunomodulatory processes showed suppression of an Ig generation in the H1R-antagonist group with enhancement in the H2R-antagonist group. The H1R-agonist group showed an increased Ig production in comparison to the H2R-agonist group. The IgM production was inhibited in the H1R-antagonist group as compared to the H2R-antagonist group, and it was also suppressed in H1R-agonist group as compared to H2R-agonist group. IgG production was inhibited in the H1R-antagonist group as opposed to the H2R-antagonist group. In contrast, the H1R-agonist group increased IgG production as compared to the H2R-agonist group. All the results were found to be statistically significant (p < 0.05 or p < 0.01). In conclusion, histamine and its receptor (H1R and H2R) agonists enhance antibody production by triggering the histamine receptors (H1R and H2R), and both the H1R antagonist and the H2R antagonist positively or negatively regulate the antibody production. The findings of this study may have clinical significance.