Anti-proliferative and immunomodulatory properties of kaffir lime leaves and bioactive compounds on macrophages co-cultured with squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide. Late-stage patients have a significant chance of local recurrence and distant metastasis, as well as poor prognosis. Therapeutic goals for patients must be improved and personalized to reduce adverse effects. This study explored the anti-proliferative activity and immunomodulation potential of the constituents of crude kaffir lime leaf extract (lupeol, citronellal and citronellol) under co-culture. Results showed high cytotoxicity to human SCC15 cell line but not to human monocyte-derived macrophages. Treatment with crude extract and the contained compounds also suppressed cell migration and colony formation of SCC15 compared to the untreated control group, while high levels of intracellular ROS production were detected in the treatment group of SCC15. The MuseTM cell analyzer revealed cell cycle arrest at G2/M phase and apoptosis induction. Inhibition of Bcl-2 and activation of Bax, leading to induction of the downstream caspase-dependent death pathway were confirmed by Western blot analysis. Co-culture with activated macrophages, kaffir lime extract and its constituents enhanced the development of pro-inflammatory (M1) macrophages and boosted TNF-α production, resulting in SCC15 apoptosis. Findings revealed novel potential activities of kaffir lime leaf extracts and their constituents in inducing M1 polarization against SCC15, as well as direct anti-proliferative activity.

Proteomic Analysis Reveals Proteins Involved in the Mode of Action of ß-Citronellol Identified From Citrus hystrix DC. Leaf Against Candida albicans.

Candida albicans is a fungus that lives primarily on the mucosal surfaces of healthy humans, such as the oral cavity, vagina, and gastrointestinal tract. This commensal organism can be controlled by other microbiota, while certain conditions can increase the risk of C. albicans outgrowth and cause disease. Prevalence of the drug-resistant phenotype, as well as the severity of C. albicans infection in immunocompromised patients, presents a challenge for scientists to develop novel, effective treatment, and prevention strategies. ß-Citronellol is an intriguing active compound of several plants that has been linked to antifungal activity, but data on the mechanism of action in terms of proteomic profiling are lacking. Here, ß-citronellol identified from Citrus hystrix DC. leaf against C. albicans were evaluated. A proteomic approach was used to identify potential target proteins involved in the mode of action of ß-citronellol. This study identified and discussed three protein groups based on the 126 major proteins that were altered in response to ß-citronellol treatment, 46 of which were downregulated and 80 of which were upregulated. Significant protein groups include cell wall proteins (e.g., Als2p, Rbt1p, and Pga4p), cellular stress response enzymes (e.g., Sod1p, Gst2p, and Ddr48p), and ATP synthesis-associated proteins (e.g., Atp3p, Atp7p, Cox1p, and Cobp). Results demonstrated the complexities of protein interactions influenced by ß-citronellol treatment and highlighted the potential of antifungal activity for future clinical and drug development research.

Oleamide-Mediated Polarization of M1 Macrophages and IL-1ß Production by Regulating NLRP3-Inflammasome Activation in Primary Human Monocyte-Derived Macrophages.

Macrophages are a type of innate immune cell that activates the NLRP3 inflammasome, causing the release of the cytokine IL-1ß, which is a crucial mediator of the inflammatory response. NLRP3 activation that is dysregulated worsens a variety of inflammatory and autoimmune diseases, as well as neurodegenerative diseases. Oleamide is an endogenous fatty acid amide that was first determined as a sleep-inducing molecule and later shown to have wide-ranging beneficial effects on the central nervous system. How oleamide influences human macrophage polarization and NLRP3-inflammasome activation remains unclear. The effect of oleamide on macrophage polarization was explored using an in vitro culture of primary human monocyte-derived macrophages (MDMs) supplemented with human serum-containing media. Cellular and molecular mechanisms of oleamide-regulated MDMs polarization were also investigated. Results showed that oleamide promoted naïve macrophages (M0) toward the M1 phenotype by upregulating M1-associated genes (IL-1ß, iNOS, CXCL10), along with downregulation of M2-associated genes (Arg-1, CD206, CCL22). Cell surface expression indicated that oleamide enhanced CD80 expression in M0 naïve macrophages and hider CD206 and CD163 expression in M2 macrophages. Higher production of IL-1ß cytokine was observed but with no alteration in IL-6 and TNF-α levels by MDMs and differentiated THP-1 models. Whether oleamide functioned as a second signal that activated the NLRP3 inflammasome and mediated IL-1ß production was further investigated using LPS-primed MDMs followed by oleamide treatment that induced activation of inflammasome-related proteins including NLRP3, ASC, cleaved casp-1, and cleaved IL-1ß. These findings suggested that oleamide promoted M1 macrophage polarization and increased IL-1ß production by activating the NLRP3 inflammasome in primary MDMs. This research reveals a new function for oleamide as well as prospective targets for treating NLRP3-related inflammatory disorders.

Phytochemicals and Immunomodulatory Effect of Nelumbo nucifera Flower Extracts on Human Macrophages.

This research characterizes phytochemicals inherent in lotus flower and investigates the antioxidant and immunomodulatory activity of ethyl acetate (EA) and ethyl alcohol (ET) lotus petal extracts. In the experiment, human monocytes-derived macrophages were stimulated by lipopoly-saccharide to mimic bacteria-induced inflammation. The results showed that ferulic acid, couma-rin, and chlorogenic acid were three dominant polyphenols. The EA and ET lotus petal extracts also possessed high antioxidant capability. Furthermore, the extracts exhibited immunomodulatory properties by suppressing TNF-α secretion in inflammatory-induced human macrophages by in-hibiting NF-κB-dependent inflammatory response. In essence, the lotus petal extracts possess reme-dial attributes beneficial to individuals afflicted with declined immune functions.

Evaluation of Anti-Inflammatory Effect of Moringa oleifera Lam. and Cyanthillium cinereum (Less) H. Rob. Lozenges in Volunteer Smokers.

Smokers have high plaque accumulation that initiates gingival inflammation and progresses to periodontitis. Thus, oral hygiene to control microbial plaque formation is an effective method of preventing gingivitis. Medicinal plants such as Moringa oleifera Lam. (MO) and Cyanthillium cinereum (Less.) H. Rob. (CC) have an anti-inflammatory effect that might improve oral health in smokers. This study evaluated the effect of MO leaf and CC extracts using MO lozenges and a combination of MO + CC lozenges on oral inflammation and gingivitis in volunteer smokers. Lozenges consisting of MO and CC extracts were developed and studied in vivo. The results showed that lozenges significantly reduced oral inflammation and gingivitis in volunteers. The gingival index (GI) of group III (MO + CC lozenges) significantly decreased, while the percentage decrease of oral inflammation in group II (MO lozenges) was significantly higher than the other groups. The percentage decrease of GI values in group II (MO lozenges) and group III (MO + CC lozenges) were significantly higher than the placebo group I. Our findings indicated that MO and MO + CC lozenges reduced oral inflammation and gingivitis and showed potential to improve oral health in smokers.

Evaluation of Mouthwash Containing Citrus hystrix DC., Moringa oleifera Lam. and Azadirachta indica A. Juss. Leaf Extracts on Dental Plaque and Gingivitis.

Oral hygiene and control of microbial plaque biofilm formation are effective methods for preventing gingivitis. Mouthwashes containing leaf extracts of the medicinal plants Citrus hystrix DC. (KL), Moringa oleifera Lam. (MO) and Azadirachta indica A. Juss. (NE) were assessed for oral healthcare and gingivitis adjunctive treatment. Three types of mouthwash were developed; KL, a combination of KL and MO (KL + MO), and a combination of KL, and NE (KL + NE). The mouthwashes were tested in vivo on 47 subjects with gingivitis who were allocated into five groups as (i) placebo, (ii) KL, (iii) KL + MO, (iv) KL + NE, and (v) 0.12% chlorhexidine gluconate (CHX). Participants were instructed to rinse with herbal mouthwash twice daily for two weeks. Gingival index (GI), plaque index (PI), and oral microbial colonies were measured at baseline and 15 days. Results showed that GI and PI of groups (ii)-(iv) significantly decreased over the placebo group, while accumulative reduction percentages of both Staphylococcus spp. and Candida spp. were found in groups (iii) and (iv). Findings indicated that the herbal mouthwashes reduced GI and PI, and showed potential as oral healthcare products.

Phytochemical Constituents of Citrus hystrix DC. Leaves Attenuate Inflammation via NF-κB Signaling and NLRP3 Inflammasome Activity in Macrophages.

Citrus hystrix DC. (CH) is found in many countries in Southeast Asia. This plant has been reported for anti-microbial, anti-cancer and anti-inflammatory bioactivities. However, the anti-inflammatory and anti-inflammasome properties of the leaves remain poorly understood. This study aimed to investigate the effect of CH leaves on NLRP3 and NF-κB signaling pathways. CH leaves were sequentially extracted using hexane, ethyl acetate and 95% ethanol to give three crude extracts. An active compound, lupeol was fractionated from the ethanolic extract using chromatographic techniques, and its structure was identified and confirmed by spectroscopic methods. Anti-inflammatory activities were observed on both lipopolysaccharide-stimulated and NLRP3 adenosine triphosphate-induced macrophages. The release of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) was analyzed by Enzyme-Linked Immunosorbent Assay (ELISA). Real-time qRT-polymerase chain reaction (PCR) was used to measure inflammatory-associated gene expression. NF-κB protein expressions were investigated using the immunoblotting technique. The active fraction of ethanolic CH leaves and lupeol significantly reduced the release of pro-inflammatory cytokines and suppressed the expression of both inflammasome genes and NF-κB proteins. The ethanolic extract of CH leaves and lupeol showed potent anti-inflammatory activities by targeting NF-κB and NLRP3 signaling pathways.

Anticancer Effect of Citrus hystrix DC. Leaf Extract and Its Bioactive Constituents Citronellol and, Citronellal on the Triple Negative Breast Cancer MDA-MB-231 Cell Line.

Triple negative breast cancer is one of the most aggressive breast cancer type with abilities of early metastasis and chemoresistance. The tropical plant Citrus hystrix DC. has been reported to promote many biological activities including anticancer. However, the effect of C. hystrix against triple negative breast cancer has not yet been identified. This study aimed to evaluate the anticancer properties of C. hystrix leaf extract and its bioactive constituents citronellol and citronellal against the triple negative breast cancer MDA-MB-231 cell line. C. hystrix leaves were powdered and sequentially macerated. The in vitro anticancer effects of C. hystrix leaf extracts, and its bioactive constituents (citronellol and citronellal) were evaluated against MDA-MB-231 cell line using cytotoxic MTT assay, cell proliferation, wound scratch migration, colony formation, cell cycle, apoptosis assay, Hoechst staining, RT-qPCR, and Western blot analysis. Results showed that crude hexane extract, citronellol, and citronellal significantly reduced cell proliferation, colony formation, and cell migration by inducing cell cycle arrest, while also inducing apoptosis in MDA-MB-231 cells through inhibition of anti-apoptotic Bcl-2 expression, leading to activation of the caspase-3-dependent pathway. This study is the first report to demonstrate the effect of C. hystrix, citronellol, and citronellal against triple negative breast cancer MDA-MB-231 cells.

In Vitro Bioassay-Guided Identification of Anticancer Properties from Moringa oleifera Lam. Leaf against the MDA-MB-231 Cell Line.

Moringa oleifera Lam. (MO) is a medicinal plant distributed across the Middle East, Asia, and Africa. MO has been used in the traditional treatment of various diseases including cancer. This study aimed to perform bioassay-guided fractionation and identification of bioactive compounds from MO leaf against MDA-MB-231 breast cancer cells. MO leaf was sequentially extracted with hexane, ethyl acetate (EtOAc), and ethanol. The most effective extract was subjected to fractionation. MO extract and its derived fractions were continuously screened for anti-cancer activities. The strongest fraction was selected for re-fractionation and identification of bioactive compounds using LC-ESI-QTOF-MS/MS analysis. The best anticancer activities were related to the fraction no. 7-derived crude EtOAc extract. This fraction significantly reduced cell viability and clonogenic growth and increased cells apoptosis. Moreover, sub-fraction no. 7.7-derived fraction no. 7 was selected for the identification of bioactive compounds. There were 10 candidate compounds tentatively identified by LC-ESI-QTOF-MS. Three of identified compounds (7-octenoic acid, oleamide, and 1-phenyl-2-pentanol) showed anticancer activities by inducing cell cycle arrest and triggering apoptosis through suppressed Bcl-2 expression which subsequently promotes activation of caspase 3, indicators for the apoptosis pathway. This study identified 10 candidate compounds that may have potential in the field of anticancer substances.

Anti-Cancer Effect of 3-Hydroxy-ß-Ionone Identified from Moringa oleifera Lam. Leaf on Human Squamous Cell Carcinoma 15 Cell Line.

Squamous cell carcinoma is the most common type of head and neck cancer worldwide. Radiation and chemotherapy are general treatments for patients; however, these remedies can have adverse side effects and tumours develop drug resistance. Effective treatments still require improvement for cancer patients. Here, we investigated the anti-cancer effect of Moringa oleifera (MO) Lam. leaf extracts and their fractions, 3-hydroxy-ß-ionone on SCC15 cell line. SCC15 were treated with and without MO leaf extracts and their fractions. MTT assay was used to determine cell viability on SCC15. Cell cycle and apoptosis were evaluated by the Muse™ Cell Analyser. Colony formation and wound closure analysis of SCC15 were performed in 6-well plates. Apoptosis markers were evaluated by immunoblotting. We found that Moringa extracts and 3-HBI significantly inhibited proliferation of SCC15. Moreover, they induced apoptosis and cell cycle arrest at G2/M phase in SCC15 compared to the untreated control. MO extracts and 3-HBI also inhibited colony formation and cell migration of SCC15. Furthermore, we observed the upregulation of cleaved caspase-3 and Bax with downregulation of anti-apoptotic Bcl-2, indicating the induction of cancer cell apoptosis. Our results revealed that MO extracts and 3-HBI provided anti-cancer properties by inhibiting progression and inducing apoptosis of SCC15.

In Vitro Antibacterial and Anti-Inflammatory Effects of Novel Insect Fungus Polycephalomyces phaothaiensis Extract and Its Constituents against Propionibacterium acnes.

Propionibacterium acnes plays an important role in the pathophysiology of acne vulgaris, the most common chronic inflammatory skin disease of the pilosebaceous unit. This study was conducted to investigate whether the entomopathogenic fungus Polycephalomyces phaothaiensis components have antibacterial and anti-inflammatory effects against P. acnes that may serve for acne treatment. A chemical study by spectroscopic analysis resulted in the identification of seven known compounds. The anti-P. acnes potency of extracts and test compounds was determined by both agar diffusion and broth dilution methods. The ethyl acetate extract from culture broth along with cordytropolone (1) and stipitalide (2) exhibited strong anti- P. acnes activity while (+)-piliformic acid (3) showed mild inhibitory activity. The anti-inflammatory effect of ethyl acetate extract and 1-3 was then examined by the quantification of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α on heat-killed P. acnes induced cytokine production by THP-1 cells. The result demonstrated that the extract and its constituents (1-3) showed a potent significant effect by inhibiting the P. acnes-induced pro-inflammatory cytokines production in THP-1. Our results suggest for the first time that P. phaothaiensis and its constituents (1 and 2) hold therapeutic value for further studies as a new alternative treatment for acne.

Molecular Identification of Phytochemical for Anticancer Treatment.

Cancer commands the second highest global mortality rate and causes severe public health problems. Recent advances have been made in cancer therapy but the incidence of the disease remains high. Research on more efficient treatment methods with reduced side effects is necessary. Historically, edible plants have been used as traditional medicines for various diseases. These demonstrate the potential of natural products as sources of bioactive compounds for anticancer treatment. Anticancer properties of phytochemicals are attributed to bioactive compounds in plant extracts that suppress cancer cell proliferation and growth by inducing both cell cycle arrest and apoptosis. This review presents a summary of the molecular identification of phytochemicals with anticancer properties and details their action mechanisms and molecular targets. Moreover, the effects of the natural product on both immunomodulatory and anticancer properties are provided.

Bioactive Compounds in Moringa oleifera Lam. Leaves Inhibit the Pro-Inflammatory Mediators in Lipopolysaccharide-Induced Human Monocyte-Derived Macrophages.

Moringa oleifera (MO) is an important plant for traditional medicine. The present study aimed to identify the MO active phytochemical compounds for their ability against inflamed macrophages. An ethyl acetate extract fraction of MO was fractionation by flash column chromatography. Human macrophages were stimulated by Lipopolysaccharide and then treated with fractions of MO to examine their anti-inflammatory activity and cellular mechanism. The active fractions were analyzed by liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometer (LC-ESI-QTOF-MS). MO treated cells showed a decreased production of pro-inflammatory mediator in response to lipopolysaccharide. This was evident at both mRNA and protein levels. The study revealed that MO suppressed mRNA expression of IL-1, IL-6, TNF-α, PTGS2, NF-κB (P50), and RelA. Furthermore, the extract effectively inhibited the expression of inflammatory mediators, including IL-6, TNF-α, and cyclooxygenase-2. Interestingly, the effect of MO inhibited phosphorylation of IκB-α and the ability to reduce expression of the nuclear factor (NF)-κB p65, suppressing its nuclear translocation. Moreover, LC-ESI-QTOF-MS analysis of the MO active fraction revealed seven compounds, namely 3,4-Methyleneazelaic acid, (2S)-2-phenylmethoxybutane-1,4-diol, (2R)-2-phenylmethoxybutane-1, 4-diol, γ-Diosphenol, 2,2,4,4-Tetramethyl-6-(1-oxobutyl)-1,3,5-cyclohexanetrione, 3-Hydroxy-ß-ionone, and Tuberonic acid. Our findings highlight the ability of MO compounds to inhibit inflammation through regulation of the NF-κB pathway.

Interaction among smoking status, single nucleotide polymorphisms and markers of systemic inflammation in healthy individuals.

Cigarette smoke contains toxic and carcinogenic substances that contribute to the development of cancer and various diseases. Genetic variation might be important, because not all smokers develop smoking-related disease. The current study addressed the possible interactions among selected single nucleotide polymorphisms (SNPs) in genes related to systemic inflammation, smoking status, the levels of circulating immune response cells and plasma biomarkers of systemic inflammation. Sixty-four healthy blood donors were recruited, 31 of whom were current smokers and 33 were never-users of tobacco products, references. Compared to references, the smokers showed significantly increased levels of circulating total white blood cells, lymphocytes, monocytes, neutrophils, basophils and C-reactive protein (CRP). Smokers also more frequently exhibited circulating cell phenotypes that are associated with an immunocompromised state: CD8dim cells in the lymphocyte group, CD13+  CD11+ , CD13+  CD14+ , CD13+  CD56+ cells in the monocyte group and CD13+  CD11+ , CD13+  CD56+ cells in the neutrophil group. We observed an interaction among SNPs, smoking status and some of the studied biomarkers. The average plasma CRP level was significantly higher among the smokers, with the highest level found among those with the CRP rs1800947 CC genotype. Additionally, an increased CD8+  GZB+ cells in the CD8dim group were found among smokers with the GZB rs8192917 AA genotype. Thus, smoking appears to be associated with systemic inflammation and increased levels of circulating immunosuppressive cells. The extent of these effects was associated with SNPs among the smokers. This observation may contribute to a better understanding of the genetic susceptibility of smoking-related disease and the variations observed in clinical outcomes.

Nosocomial infection of multidrug-resistant Acinetobacter baumannii in Thailand.

Infections caused by Acinetobacter baumannii have become a critical problem for hospital patients worldwide. We investigated the multidrug-resistant A baumannii strain that caused hospital-acquired infections in Uthai Thani Hospital, Thailand, between 2006 and 2014. In summary, the spread of a clonally related multidrug-resistant A baumannii strain was the primary cause of nosocomial infections in Uthai Thani Hospital.

Kaffir lime leaves extract inhibits biofilm formation by Streptococcus mutans.

OBJECTIVES: Although kaffir lime has been reported to exhibit antioxidant and antileukemic activity, little is known about the antimicrobial effect of kaffir lime extract. Because Streptococcus mutans has been known to cause biofilm formation, it has been considered the most important causative pathogen of dental caries. Thus, the effective control of its effects on the oral biofilm is the key to the prevention of dental caries. The aims of the present study were to investigate the effect of kaffir lime leaves extract on biofilm formation and its antibacterial activity on S. mutans. METHODS: We examined the effect of kaffir lime leaves extract on growth and biofilm formation of S. mutans. For the investigation we used a kaffir lime extract with high phenolic content. The minimum inhibitory concentration of the extract was determined by broth microdilution assay. The inhibitory effect of the test substances on biofilm formation was also investigated by biofilm formation assay and qRT-PCR of biofilm formation-associated genes. RESULTS: Kaffir lime leaves extract inhibits the growth of S. mutans, corresponding to the activity of an antibiotic, ampicillin. Formation of biofilm by S. mutans was also inhibited by the extract. These results were confirmed by the down-regulation of genes associated with the biofilm formation. CONCLUSIONS: The findings highlight the ability of kaffir lime leaves extract to inhibit S. mutans activity, which may be beneficial in the prevention of biofilm formation on dental surface, reducing dental plaque and decreasing the chance of dental carries.

Protein kinase cα regulates the expression of complement receptor Ig in human monocyte-derived macrophages.

The complement receptor Ig (CRIg) is selectively expressed by macrophages. This receptor not only promotes the rapid phagocytosis of bacteria by macrophages but also has anti-inflammatory and immunosuppressive functions. Previous findings have suggested that protein kinase C (PKC) may be involved in the regulation of CRIg expression in human macrophages. We have now examined the role of PKCα in CRIg expression in human monocyte-derived macrophages (MDM). Macrophages nucleofected with plasmid containing short hairpin RNA against PKCα showed markedly reduced expression of PKCα, but normal PKCζ expression, by Western blotting analysis, and vice versa. PKCα-deficient MDM showed increased expression of CRIg mRNA and protein (both the long and short form), an increase in phagocytosis of complement-opsonized Candida albicans, and decreased production of TNF-α and IL-6. TNF-α caused a marked decrease in CRIg expression, and addition of anti-TNF mAb to the TNF-α-producing MDMs increased CRIg expression. PKCα-deficient macrophages also showed significantly less bacterial LPS-induced downregulation of CRIg. In contrast, cells deficient in PKCα showed decreased expression of CR type 3 (CR3) and decreased production of TNF-α and IL-6 in response to LPS. MDM developed under conditions that increased expression of CRIg over CR3 showed significantly reduced production of TNF-α in response to opsonized C. albicans. The findings indicate that PKCα promotes the downregulation of CRIg and upregulation of CR3 expression and TNF-α and IL-6 production, a mechanism that may promote inflammation.

An ethyl acetate fraction of Moringa oleifera Lam. Inhibits human macrophage cytokine production induced by cigarette smoke.

Moringa oleifera Lam. (MO) has been reported to harbor anti-oxidation and anti-inflammatory activity and useful in the treatment of inflammatory diseases. However, despite these findings there has been little work done on the effects of MO on immune cellular function. Since macrophages, TNF and related cytokines play an important pathophysiologic role in lung damage induced by cigarette smoke, we examined the effects of MO on cigarette smoke extract (CSE)-induced cytokine production by human macrophages. An ethyl acetate fraction of MO (MOEF) was prepared from fresh leaves extract of Moringa and shown to consist of high levels of phenolic and antioxidant activities. Human monocyte derived macrophages (MDM) pre-treated with varying concentrations of MOEF showed decreased production of TNF, IL-6 and IL-8 in response to both LPS and CSE. The decrease was evident at both cytokine protein and mRNA levels. Furthermore, the extract inhibited the expression of RelA, a gene implicated in the NF-κB p65 signaling in inflammation. The findings highlight the ability of MOEF to inhibit cytokines (IL-8) which promote the infiltration of neutrophils into the lungs and others (TNF, IL-6) which mediate tissue disease and damage.

Murine monoclonal antibodies neutralizing the cytotoxic activity of diphtheria toxin.

In this study, murine monoclonal antibodies that specifically bound to the A and B subunits of diphtheria toxin (DT) were produced by conventional hybridoma technology using the spleens of BALB/c mice immunized with diphtheria DTP vaccine and CRM197. Monoclonal antibodies specific to the A subunit, i.e. clone AC5, as well as those specific to the B subunit, i.e. clone BB7, could neutralize the DT-mediated cytotoxicity to Vero cells in microcultures. The DT neutralizing mechanisms have yet to be determined. The MAbBB7 is hypothesized to either interfere with the DT receptor binding or with the pore forming function of the T domain of the B subunit. The MAbAC5 could neutralize the DT mediated cytotoxicity when mixed with the DT before adding to the Vero cell culture thus suggesting that the antibody interfered with the translocation of the A subunit. The A subunit-antibody complex might be too large to pass through the membrane channel formed by the T domain and thus prevent the accessibility of the A subunit to the cytosolic target. It is also possible that the MAb AC5 blocked the enzymatic active site of the enzyme catalytic subunit. While further experiments are needed to localize the epitopes of the two MAbs on the holo-DT in order to reveal the DT neutralizing mechanisms, both MAbs in their humanized forms have a high potential as human therapeutic antibodies for diphtheria.