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.     

Heterosubtypic immunity to influenza mediated by liposome adjuvanted H5N1 recombinant protein vaccines

A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP + M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8⁺, intracellular IFNγ⁺ cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans.     

Concordance of skin prick test and serum-specific IgE to locally produced component-resolved diagnostics for cockroach allergy

Background

Diagnosis of Periplaneta americana (American cockroach, ACR) allergy is commonly performed based on clinical history and skin prick test (SPT) or specific serum IgE (sIgE) measurement. The concordance of the findings with the SPT and sIgE results has never been investigated.

Interleukin-25 (IL-25) Promotes Efficient Protective Immunity against Trichinella spiralis Infection by Enhancing the Antigen-Specific IL-9 Response

Mammalian hosts often develop distinct immune response against the diverse parasitic helminths that have evolved for immune evasion. Interleukin-25 (IL-25), an IL-17 cytokine family member, plays a key role in initiating the protective immunity against several parasitic helminths; however, the involvement and underlying mechanisms by which IL-25 mediates immune response against Trichinella spiralis infection have not been investigated. Here we showed that IL-25 functions in promoting protective immunity against T. spiralis infection. Mice treated with IL-25 exhibited a lower worm burden and fewer muscle larvae in the later stage of T. spiralis infection. In contrast, mice treated with neutralizing antibody against IL-25 failed to expel T. spiralis effectively. During T. spiralis infection, intestinal IL-25 expression was rapidly elevated before the onset of IL-4 and IL-9 induction. While antigen-specific Th2 and Th9 immune responses were both developed during T. spiralis infection, an antigen-specific Th9 response appeared to be transiently induced in the early stage of infection. Mice into which antigen-specific T cells deficient in IL-9 were transferred were less effective in worm clearance than those given wild-type T cells. The strength of the antigen-specific Th9 immune response against T. spiralis could be enhanced or attenuated after treatment with IL-25 or neutralizing antibody against IL-25, respectively, correlating positively with the levels of intestinal mastocytosis and the expression of IL-9-regulated genes, including mast cell- and Paneth cell-specific genes. Thus, our study demonstrates that intestinal IL-25 promotes protective immunity against T. spiralis infection by inducing antigen-specific Th9 immune response.     

Elderly blacks have a blunted sympathetic neural responsiveness but greater pressor response to orthostasis than elderly whites

Neural control of blood pressure (BP) has been reported to differ between young blacks and whites. We hypothesized that elderly blacks have enhanced sympathetic neural responses during orthostasis compared with elderly whites. Muscle sympathetic nerve activity, arm-cuff BP, and heart rate were recorded continuously, and cardiac output, stroke volume, and total peripheral resistance were measured intermittently during supine and 5-minute 60° upright tilt in 10 blacks (65 [SD, 4] years; 4 women) and 20 whites (68 [6] years; 8 women). We found that muscle sympathetic nerve activity burst frequency was similar between blacks and whites in the supine position (44 [10] versus 42 [7] bursts per minute) and during upright tilt (59 [11] versus 60 [9] bursts per minute; P=0.846 for race, P<0.001 for posture, and P=0.622 for interaction). However, upright total muscle sympathetic nerve activity was smaller in blacks than in whites (162 [39] versus 243 [112]%; P=0.003). Systolic BP, heart rate, cardiac output, and stroke volume were not different between groups. Diastolic BP was similar in the supine position, increased in all of the subjects during tilting; upright diastolic BP was greater in blacks than in whites (80 [10] versus 71 [7] mmHg; P=0.008). Total peripheral resistance did not differ between blacks and whites in the supine position or during upright tilt (P=0.354 for race, P<0.001 for posture, P=0.825 for interaction). Thus, elderly blacks have a blunted sympathetic neural responsiveness but enhanced pressor response to orthostasis compared with elderly whites, which may be attributable to an augmented sympathetic vascular transduction and/or nonadrenergic vasoconstrictor mechanisms (ie, angiotensin II or the venoarteriolar response).     

Spironolactone prevents chlorthalidone-induced sympathetic activation and insulin resistance in hypertensive patients

Recent studies from our laboratory indicate that chlorthalidone triggers persistent activation of the sympathetic nervous system and promotes insulin resistance in hypertensive patients, independent of serum potassium. Mechanisms underlying these adverse effects of chlorthalidone remain unknown, but increasing evidence in rodents suggests the role of angiotensin and aldosterone excess in inducing both sympathetic overactivity and insulin resistance. Accordingly, we conducted studies in 17 subjects with untreated stage 1 hypertension, measuring sympathetic nerve activity at baseline and after 12 weeks of chlorthalidone alone (25 mg/d), chlorthalidone plus spironolactone, and chlorthalidone plus irbesartan, using randomized crossover design. We found that chlorthalidone alone decreased 24-hour ambulatory blood pressure from 135±3/84±2 to 124±2/78±2 mm Hg and significantly increased sympathetic nerve activity from baseline (from 41±3 versus 49±4 bursts per minute; P<0.01). The addition of spironolactone to chlorthalidone returned sympathetic nerve activity value to baseline (42±3 bursts per minute; P>0.05), whereas the addition of irbesartan failed to alter the sympathetic nerve activity response to chlorthalidone in the same subjects (52±2 bursts per minute; P<0.01) despite a similar reduction in ambulatory blood pressure (121±2/75±2 and 121±2/75±2 mm Hg, respectively). Chlorthalidone alone also increased indices of insulin resistance, which was not observed when used in combination with spironolactone. In conclusion, our study demonstrates beneficial effects of spironolactone in attenuating both chlorthalidone-induced sympathetic activation and insulin resistance in humans, independent of blood pressure reduction. Because sympathetic overactivity and insulin resistance contribute to the poor prognosis in patients with cardiovascular disease, combination therapy of chlorthalidone with mineralocorticoid receptor antagonists may constitute a preferable regimen than chlorthalidone alone in hypertensive patients.     

Monoclonal antibody that neutralizes pertussis toxin activities

Pertussis or whooping cough is a disease with high mortality among infants and small children. The disease is caused by infection of the respiratory tract by a gram negative bacterium, Bordetella pertussis. The superficial colonized bacteria produce a myriad of toxins which enter the circulation causing various pathophysiologicalal changes in the host. Although antimicrobial therapy reduces the number of the coughed out bacteria and also the infectious time of the infected host, but it is not effective in amelioration of the clinical manifestations as the pertussis morbidity is due principally to the pertussis toxin (PT). Antibody based-therapy is frequently practiced in conjunction with other supportive measure to resuscitate the patient. Nevertheless, human derived antiserum against PT is of the limited supply and the ethical concern. Thus in this study a hybridoma clone, i.e. clone PT6-2G6, secreting monoclonal antibody (MAb) specific to the S1 subunit, the active enzyme of the PT that intracellularly ADP-ribosylates the host Gi-protein, was produced. The MAbPT6-2G6 inhibited the in vitro hemagglutination of chicken erythrocytes which is the activity of the B oligomer of PT; thus we hypothesize that the MAb bound to its epitope on the S1 subunit and stereologically hinders the binding sites of the B subunits. The MAb also inhibited ex vivo Chinese hamster ovarian cell clustering and neutralized the in vivo leucocytosis- promotion in mice which are usually mediated by intracellular S1 subunit. The large molecular nature of the intact MAb and its molecular hydrophilicity led us to speculate that the observed PT neutralizing activities of the MAb were due to interfering with the cellular entry of the S1 rather than the intracellular enzyme neutralizing activity per se. While further experiments are needed to pinpoint the MAb neutralizing activity and to identify the amino acid sequence and location of the MAbPT6-2G6 epitope, our findings indicate that this murine MAb, in its humanized-version, should have high therapeutic potential for pertussis.     

Native troponin-T of the American cockroach (CR), Periplaneta americana, binds to IgE in sera of CR allergic Thais

The American cockroach, Periplaneta americana, is the predominant cockroach (CR) species in Thailand and a major source of indoor allergens second only to the house dust mite. The incidence of CR allergy among allergic Thai patients is increasing but basic information on the allergenic components is scarce. In this study a recombinant troponin-T was produced by using cDNA prepared from RNA of the P. americana as a template and PCR primers designed from the P. americana troponin-T sequence deposited in the GenBank database. The recombinant protein (Mr approximately 50) did not bind to IgE in the sera of 18 skin prick test positive CR allergic patients. Rabbit polyclonal antiserum (PAb) against the recombinant troponin-T was produced and used in preparing an affinity column for the purification of native troponin-T from the crude P. americana extract (Mr approximately 47). IgE-immunoblotting revealed that the native protein bound to IgE in 3 of the 18 (16.7%) patients. Our results imply that native P. americana troponin-T, but not its recombinant counterpart, is a minor allergen among the CR allergic Thais.