Dopamine β-hydroxylase deficiency impairs cellular immunity

Norepinephrine, released from sympathetic neurons, and epinephrine, released from the adrenal medulla, participate in a number of physiological processes including those that facilitate adaptation to stressful conditions. The thymus, spleen, and lymph nodes are richly innervated by the sympathetic nervous system, and catecholamines are thought to modulate the immune response. However, the importance of this modulatory role in vivo remains uncertain. We addressed this question genetically by using mice that lack dopamine β-hydroxylase (dbh−/− mice). dbh−/− mice cannot produce norepinephrine or epinephrine, but produce dopamine instead. When housed in specific pathogen-free conditions, dbh−/− mice had normal numbers of blood leukocytes, and normal T and B cell development and in vitro function. However, when challenged in vivo by infection with the intracellular pathogens Listeria monocytogenes or Mycobacterium tuberculosis, dbh−/− mice were more susceptible to infection, exhibited extreme thymic involution, and had impaired T cell function, including Th1 cytokine production. When immunized with trinitrophenyl-keyhole limpet hemocyanin, dbh−/− mice produced less Th1 cytokine-dependent-IgG2a antitrinitrophenyl antibody. These results indicate that physiological catecholamine production is not required for normal development of the immune system, but plays an important role in the modulation of T cell-mediated immunity to infection and immunization.

A second-generation vaccine protects against the psychoactive effects of cocaine

The effects of immunization with the second-generation cocaine immunoconjugate GND-keyhole limpet hemocyanin (KLH) or with the anti-cocaine mAb GNC92H2 were assessed in a model of acute cocaine-induced locomotor activity. After i.p. administration of cocaine⋅HCl (15 mg/kg), rats were tested in photocell cages, and stereotypy was rated to determine preimmunization drug response (baseline). Experimental animals were subjected to an immunization protocol with GND-KLH or treated with the mAb GNC92H2. Rats were then challenged with systemic cocaine, and their locomotor responses were again measured. Active immunization with GND-KLH produced a 76% decrease in the ambulatory measure (crossovers) in the experimental group and a 12% increase in the control group compared with baseline values. Also, stereotypic behavior was significantly suppressed in the vaccinated animals. Decreases in both measures were seen in the experimental group on two subsequent challenges. The maximum effect was observed at the time of the second challenge with a dramatic 80% decrease in crossovers. Treatment with GNC92H2 resulted in a 69% decrease in crossovers compared with baseline. This effect persisted across two additional challenges over 11 days with decreases of 46–47%. In contrast, the control group showed increases of up to 28%. Significant differences between groups were observed in the stereotypic measure in all three challenges. The results indicate that these immunopharmacotherapeutic agents have significant cocaine-blockade potential and therefore may offer an effective strategy for the treatment of cocaine abuse.

Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewisy conjugates in mice

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewisy (Ley), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Ley as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Ley conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Ley-serine epitopes and the Pam3Cys immunostimulating moiety was found to be superior to a similar construct containing only one Ley-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Ley-serine Pam3Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.

S-Adenosyl-l-Methionine:l-Methionine S-Methyltransferase from Germinating Barley1 : Purification and Localization

S-Adenosyl-l-methionine:l-methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-l-methionine (SMM) from l-methionine and S-adenosyl-l-methionine. SMM content increases during barley (Hordeum vulgare L.) germination. Elucidating the role of this compound is important from both a fundamental and a technological standpoint, because SMM is the precursor of dimethylsulfide, a biogenic source of atmospheric S and an undesired component in beer. We present a simple purification scheme for the MMT from barley consisting of 10% to 25% polyethylene glycol fractionation, anion-exchange chromatography on diethylaminoethyl-Sepharose, and affinity chromatography on adenosine-agarose. A final activity yield of 23% and a 2765-fold purification factor were obtained. After digestion of the protein with protease, the amino acid sequence of a major peptide was determined and used to produce a synthetic peptide. A polyclonal antibody was raised against this synthetic peptide conjugated to activated keyhole limpet hemocyanin. The antibody recognized the 115-kD denatured MMT protein and native MMT. During barley germination, both the specific activity and the amount of MMT protein increased. MMT-specific activity was found to be higher in the root and shoot than in the endosperm. MMT could be localized by an immunohistochemical approach in the shoot, scutellum, and aleurone cells but not in the root or endosperm (including aleurone).

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

Tick saliva inhibits the chemotactic function of MIP-1 alpha and selectively impairs chemotaxis of immature dendritic cells by down-regulating cell-surface CCR5

Ticks are blood-feeding arthropods that secrete immunomodulatory molecules through their saliva to antagonize host inflammatory and immune responses. As dendritic cells (DCs) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC migration and function. Bone marrow-derived immature DCs pre-exposed to tick saliva showed reduced migration towards macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta and regulated upon activation, normal T cell expressed and secreted (RANTES) chemokines in a Boyden microchamber assay. This inhibition was mediated by saliva which significantly reduced the percentage and the average cell-surface expression of CC chemokine receptor CCR5. In contrast, saliva did not alter migration of DCs towards MIP-3 beta, not even if the cells were induced for maturation. Next, we evaluated the effect of tick saliva on the activity of chemokines related to DC migration and showed that tick saliva per se inhibits the chemotactic function of MIP-1 alpha, while it did not affect RANTES, MIP-1 beta and MIP-3 beta. These data suggest that saliva possibly reduces immature DC migration, while mature DC chemotaxis remains unaffected. In support of this, we have analyzed the percentage of DCs on mice 48 h after intradermal inoculation with saliva and found that the DC turnover in the skin was reduced compared with controls. Finally, to test the biological activity of the saliva-exposed DCs, we transferred DCs pre-cultured with saliva and loaded with the keyhole limpet haemocyanin (KLH) antigen to mice and measured their capacity to induce specific T cell cytokines. Data showed that saliva reduced the synthesis of both T helper (Th)1 and Th2 cytokines, suggesting the induction of a non-polarised T cell response. These findings propose that the inhibition of DCs migratory ability and function may be a relevant mechanism used by ticks to subvert the immune response of the host. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Acute schistosomiasis: clinical, diagnostic and therapeutic features

Three distinct syndromes caused by schistosomiasis have been described: cercarial dermatitis or swimmer's itch, acute schistosomiasis or Katayama fever, and chronic schistosomiasis. Complications of acute schistosomiasis have also been reported. The absence of a serological marker for the acute stage has hindered early diagnosis and treatment. Recently, an ELISA test using KLH (keyhole limpet haemocyanin) as antigen, has proved useful in differentiating acute from chronic schistosomiasis mansoni. Clinical and experimental evidence indicate that steroids act synergistically with schistosomicides in the treatment of Katayama syndrome. In this paper, clinical, diagnostic and therapeutic features of acute schistosomiasis are updated.

GP38, P28-I and P28-II: candidates for a vaccine against Schistosomiasis

Three antigens protective against Schistosoma mansoni have been extensively characterized. The schistosomulum surface antigen GP38 possesses an immunodominant carbohydrate epitope of which the structure has been defined. Protection can be achieved via the transfer of monoclonal antibodies recognizing the epitope or by immunization with anti-idiotype monoclonal antibodies. The glycan epitope is shared with the intermediate host, Biomphalaria glabrata as well as being present on other molluscs, including the Keyhole Limpet. A group of molecules at 28 kDa were initially characterized in adult worms and shown to protect rats and mice against a challenge infection. One of these molecules, P28-I, was cloned and expressed in E. coli, yeast and vaccinia virus. The recombinant antigen significantly protected rats, hamsters and baboons against a challenge infection. P28-I is a glutathione-S-transferase and the recombinant antigen produced in yeast exhibits the enzyme activity and has been purified to homogeneity by affinity chromatography. A second P28 antigen, P28-II, has also been cloned, fully sequenced and expressed. This recombinant antigen also protects against S. mansoni infection.

Acute human schistosomiasis mansoni

The acute schistosomiasis is the toxemic disease that follow the Schistosoma cercariae active penetration trough screen in the immunologicaly naive vertebrate host. The clinical picture starts two to eight weeks after the first contact with the contaminated water. Susceptible patients present a syndrome comprising fever, diarrhea, toxemia and hepatosplenomegaly. Diagnosis is based on epidemiological and clinical features, presence of Schistosoma eggs in the feces, enlargement of abdominal lymph nodes by ultrasonography and by detection of high antibodies levels against the antigen keyhole limpet haemocyanin. Different rates of cure have been observed with specific medication and for the most severe clinical presentations the use of steroids reduces the systemic and allergic manifestations.

The serological differentiation of acute and chronic schistosomiasis japonica using IgA antibody to egg antigen

Two groups of Schistosoma japonicum infected patients (acute and chronic ) and non-infected individuals were studied using IgA antibody to egg antigen (SEA) and IgG and IgM antibodies to keyhole limpet haemocyanin (KLH). The means and standard deviation of the optical density in ELISA of acute, chronic and negative groups for IgA anti-SEA were 583±124.7, 98.2±78.8 and 82.2±39. 3, respectively. There was a statistically significance between acute patients and chronic patients (P<0.01). The means and standard deviation of IgG and IgM antibodies to KLH were 501.5±150.6, 113.0±79.1, 28.8±56.3 and 413.6±148.5, 70.2±14.8, 65.3±45.3, respectively. The detection results of IgA to SEA compared with the IgG and IgM to KLH did not demonstrate a significant difference (P>0.01). The sensitivities of IgA to SEA and IgG and IgM antibodies to KLH for the detection of acute infection were 95.24%, 90.48% and 85.71%, respectively. Therefore, this study showed that the detection of IgA to SEA is also a useful new method for the serological differentiation of acute and chronic schistosomiasis japonica in humans.

Developmental, metabolic and immunological costs of flea infestation in the common vole

Parasites use resources from their hosts, which can indirectly affect a number of host functions because of trade-offs in resource allocation. In order to get a comprehensive view of the costs imposed by blood sucking parasites to their hosts, it is important to monitor multiple components of the development and physiology of parasitized hosts over long time periods. The effect of infestation by fleas on body mass, body length growth, haematocrit, resistance to oxidative stress, resting metabolic rate and humoral immune response were experimentally evaluated. During a 3-month period, male common voles, Microtus arvalis, were either parasitized by rat fleas (Nosopsyllus fasciatus), which are naturally occurring generalist ectoparasites of voles, or reared without fleas. Then voles were challenged twice by injecting Keyhole Limpet Haemocyanin (KLH) to assess whether the presence of fleas affects the ability of voles to produce antibodies against a novel antigen. During the immune challenge we measured the evolution of body mass, haematocrit, resistance to oxidative stress and antibody production. Flea infestation negatively influenced the growth of voles. Moreover, parasitized voles had reduced haematocrit, higher resting metabolic rate and lower production of antibodies against the KLH. Resistance to oxidative stress was not influenced by the presence of fleas. During the immune challenge with KLH, body mass decreased in both groups, while the resistance to oxidative stress remained stable. In contrast, the haematocrit decreased only in parasitized voles. Our experiment shows that infestation by a haematophageous parasite negatively affects multiple traits like growth, energy consumption and immune response. Fleas may severely reduce the survival probability and reproductive success of their host in natural conditions.

Longevity differs among sexes but is not affected by repeated immune activation in voles (Microtus arvalis)

Investment of resources in immune defences, despite obvious short-term benefits, may be detrimental to long-term maintenance and thus decrease longevity in absence of parasites. In addition, females and males may differ in immune investment and intrinsic longevity because they are subjected to different degrees of sexual competition and extrinsic mortality. In order to test if sex-specific investment in mounting an immune response reduced longevity, we compared the longevity of captive male and female common voles Microtus arvalis regularly challenged with keyhole limpet haemocyanin, an antigen which elicits the production of antibodies, to the longevity of voles injected with the corresponding antigen-free buffer (phosphate-buffered saline). Injections were repeated every 28 days to mimic a chronic infection. The magnitude of immune response did not vary between males and females and did not affect longevity. Overall, females lived longer than males, independently of the immune challenge. Thus, the long-term costs of immunity seem small in voles. The longevity pattern is consistent with the prediction that male-biased predation or parasitism in the wild causes reduced intrinsic lifespan, but this reduction is not mediated by a decrease in male immunity

Keyhole sign: how specific is it for the diagnosis of posterior urethral valves?

Objectives Posterior urethral valves (PUV) are the most common cause of renal impairment in boys during early childhood. Although antenatal suspicion of this pathology has become quite common in recent years, prenatal diagnosis remains challenging. The aim of this study was to evaluate the predictive value of different ultrasound criteria currently used to diagnose PUV. Methods We reviewed the antenatal and postnatal files of 54 male patients referred to our center from 2000 to 2006 after detection of fetal bilateral hydronephrosis. The following ultrasound criteria were evaluated in relation to the postnatal diagnosis of P U V: amniotic fluid volume, bladder wall thickness, bladder dilatation and the presence of the `keyhole sign`. Results Forty-two fetuses (77.8%) were suspected to have PUV on prenatal examination. Out of these, 29 (69.0%) had PUV confirmed postnatally. The sensitivity and specificity of the antenatal diagnosis of PUV were 94% and 43%, respectively. Increased bladder wall thickness and bladder dilatation were highly associated with the diagnosis of PUV (P < 0.001). However, a thick-walled bladder was observed in 39.1% and a dilated bladder in 47.8% of the infants with a postnatal diagnosis other than PUV. The presence of the keyhole sign was not found to predict a diagnosis of PUV (P = 0.27). Conclusion In this series the use of classical prenatal ultrasound signs to diagnose PUV showed high sensitivity but low specificity. The best diagnostic indicators were increased bladder wall thickness and dilatation of the bladder. The keyhole sign was not found to be a reliable predictor of PUV. Copyright (C) 2009 ISUOG. Published by John Wiley & Sons, Ltd.