Significant Levels Of IgG Research Articles

Immunogenicity of a pentavalent recombinant Escherichiacoli bacterin against enterotoxemia and botulism in sheep

IntroductionProducing commercial bacterins/toxoids against Clostridium spp. is laborious and hazardous. Conversely, developing prototype vaccines using purified recombinant toxoids, though safe and effective, is both laborious and costly for application in production animals. ObjectiveConsidering that inactivated recombinant Escherichiacoli (bacterin) is a simple, cost-effective, and to be safe solution, we evaluated, for the first time, a pentavalent formulation of recombinant bacterins containing the alpha, beta, and epsilon toxins of Clostridiumperfringens and C and D neurotoxins of Clostridiumbotulinum in sheep. MethodsSubcutaneously, 18 Texel sheep received two doses (200 μg of each antigen) of recombinant bacterin (n = 7) or purified recombinant antigens (n = 6) on days 0 and 28, while the control group (n = 5) did not receive an immunization. Sera samples from days 0 (before the 1st dose), 28 (before the 2nd dose), and 56, 84, and 112 were used for measuring IgG (indirect ELISA) and neutralizing antibodies (mouse serum neutralization). ResultsBoth formulations induced significant levels of IgG against all five toxins (p < 0.05) up to day 112, with peaks at days 28 and 56 post-immunization. The expected booster effect occurred only for the botulinum toxins. The neutralizing antibody titers were satisfactory against ETX (≥2 IU/ml for both formulations) and BoNT-D [5 IU/ml (bacterin) and 10 IU/ml (purified)]. ConclusionWhile adjustments are required, the recombinant bacterin platform holds great potential for polyvalent vaccines due to its straightforward, safe, and cost-effective production, establishing it as a user-friendly technology for the veterinary immunobiological industry.

In silico design of multi-epitope vaccines against the hantaviruses by integrated structural vaccinology and molecular modeling approaches.

Hantaviruses are single-stranded RNA viruses belonging to the family Bunyaviridae that causes hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) worldwide. Currently, there is no effective vaccination or therapy available for the treatment of hantavirus, hence there is a dire need for research to formulate therapeutics for the disease. Computational vaccine designing is currently a highly accurate, time and cost-effective approach for designing effective vaccines against different diseases. In the current study, we shortlisted highly antigenic proteins i.e., envelope, and nucleoprotein from the proteome of hantavirus and subjected to the selection of highly antigenic epitopes to design of next-generation multi-epitope vaccine constructs. A highly antigenic and stable adjuvant was attached to the immune epitopes (T-cell, B-cell, and HTL) to design Env-Vac, NP-Vac, and Com-Vac constructs, which exhibit stronger antigenic, non-allergenic, and favorable physiochemical properties. Moreover, the 3D structures were predicted and docking analysis revealed robust interactions with the human Toll-like receptor 3 (TLR3) to initiate the immune cascade. The total free energy calculated for Env-Vac, NP-Vac, and Com-Vac was -50.02 kcal/mol, -24.13 kcal/mol, and -62.30 kcal/mol, respectively. In silico cloning, results demonstrated a CAI value for the Env-Vac, NP-Vac, and Com-Vac of 0.957, 0.954, and 0.956, respectively, while their corresponding GC contents were 65.1%, 64.0%, and 63.6%. In addition, the immune simulation results from three doses of shots released significant levels of IgG, IgM, interleukins, and cytokines, as well as antigen clearance over time, after receiving the vaccine and two booster doses. Our vaccines against Hantavirus were found to be highly immunogenic, inducing a robust immune response that demands experimental validation for clinical usage.

Potato apyrase reduces granulomatous area and increases presence of multinucleated giant cells in murine schistosomiasis.

Granulomas are inflammatory tissue responses directed to a set of antigens. Trapped Schistosoma mansoni eggs promote productive granulomas in the tissues, and they are the main damage caused by schistosomiasis. Some S. mansoni antigenic proteins may have a direct involvement in the resolution of the granulomatous response. The ATP diphosphohydrolases isoforms of this parasite are immunogenic, expressed in all phases of the parasite life cycle and secreted by eggs and adult worms. Potato apyrase is a vegetable protein that cross-reactive with parasite ATP diphosphohydrolases isoforms. In this study, the vegetable protein was purified, before being inoculated in C57BL/6 mice that were later infected with cercariae. Sixty days after infection, adult worms were recovered, antibodies and cytokines were measured, and morphological granuloma alterations evaluated. Immunization of the animals induced significant levels of IgG and IgG1 antibodies and IFN-γ, IL-10 and IL-5 cytokines, but not IL-13, suggesting that potato apyrase is an immunoregulatory protein. Supporting this hypothesis, it was found that liver damage associated with schistosomiasis was mitigated, reducing the size of the areas affected by granuloma to 35% and increasing the presence of multinucleated giant cells in this environment. In conclusion, potato apyrase was found to be effective immunomodulatory antigen for murine schistosomiasis.

Association Between Secondary Botulinum ToxinA Treatment Failure in Cosmetic Indication and Anti-Complexing Protein Antibody Production.

IntroductionBotulinum toxin A (BoT/A) treatment failure (BTF) affects patients subjected to repeated BoT/A exposure for cosmetic indications. BoT/A’s general formulation contains core BoT/A and complexing proteins. BTF may be caused by antibody-induced treatment failure. Antibodies against core BoT/A can occur; however, anti-complexing protein antibodies have never been demonstrated, and tools for anti-complexing protein antibody detection have not been developed. The aim of this study was to evaluate immune involvement in BoT/A-nonresponsive patients.MethodsPatients suspected of nonresponsiveness to BoT/A for cosmetic indications were recruited. All volunteers were categorized as BoT/A-responsive or BoT/A-tolerant according to frontalis testing with onabotulinumtoxinA (onaA). Twenty-two BoT/A-tolerant volunteers were recruited separately for frontalis testing with incobotulinumtoxinA (incoA). Anti-BoT/A and anti-complexing protein antibodies were quantified by special ELISA using sera from blood sampled before and after frontalis testing.ResultsSignificantly higher levels of IgG against complexing protein were detected in onaA-tolerant sera but not in onaA-responders, leading to proposals that anti-complexing protein antibodies could cause onaA unresponsiveness. Some onaA-tolerant patients according to frontalis test with incoA were responsive to incoA. Newly developed absorption ELISA confirmed that incoA-responsive sera predominantly contained IgG against complexing proteins, whereas incoA-tolerant sera contained significant levels of IgG against core BoT/A. The presence of anti-complexing protein antibodies higher than 90.75% in sera of onaA-tolerant patients could respond to incoA. The ELISA technique might be employed as a tool to predict incoA responsiveness. Our frontalis testing after incoA treatment showed that anti-incoA IgG levels were not increased by incoA.ConclusionsBoT/A-exposed patients may develop antibodies against core botulinum toxin and complexing proteins. Our study is the first to demonstrate that anti-complexing protein antibodies cause BTF. High levels of antibodies against complexing proteins can cause onaA unresponsiveness, although some patients were still incoA-responsive. Our developed ELISA to detect anti-complexing protein antibodies can determine whether onaA-tolerant patients respond to incoA without incoA frontalis testing.

A nanoemulsion-adjuvanted intranasal H5N1 influenza vaccine protects ferrets against homologous and heterologous H5N1 lethal challenge

BackgroundFlu vaccines administered intramuscularly (IM) have shown seasonally fluctuating efficacy, 20–60%, throughout the last 15 years. We formulated a recombinant H5 (rH5) in our Nanovax® (NE01) (rH5/NE01) adjuvant for intranasal vaccination in ferrets. We evaluated the regimen, one vs two immunization, and cross clade protection a ferret challenge model. MethodsPlant derived recombinant H5 (rH5) antigen was formulated with NE01 and administered intranasally to ferrets. Immunogenicity (IgG), hemagglutination inhibition (HI), and protection against lethal challenge, were measured following one or two immunizations. Protection against homologous (strain A/Indo) and heterologous (strain A/Vn) was evaluated in ferrets following two immunizations. ResultsIN immunization with rH5/NE01 induced significant IgG levels after one and two immunizations. One vaccination did not induce any HI while low HI was measured after two immunizations. Homologous challenge with H5N1 A/ Indonesia showed 100% survival, with minimal weight loss in animals vaccinated twice compared to the unvaccinated controls. Analysis of nasal wash from these challenged ferrets vaccinated twice showed decreased viral shedding compared to unvaccinated controls. Interestingly, animals that received one vaccination showed 88% survival with moderate weight loss. Cross clade protection was evaluated using an increased antigen dose (45 µg rH5). Vaccinated animals demonstrated increased IgG and HAI antibody responses. Both homologous (A/Indo) and heterologous challenge (A/Vietnam) following two immunizations showed 100% survival with no loss of body weight. However viral clearance was more rapid against the homologous (day 3) compared to the heterologous (day 5) post challenge. ConclusionIntranasal administration of NE01 adjuvant-formulated rH5 vaccine elicited systemic and probably mucosal immunity that conferred protection against lethal challenge with homologous or heterologous viral strains. It also enhanced viral clearance with decreased shedding. These outcomes strongly suggest that intranasal immunization using NE01 against flu infections warrants clinical testing.

Recombinant BCG strains expressing chimeric proteins derived from Leptospira protect hamsters against leptospirosis

Leptospirosis is a zoonosis that is responsible for one million human cases per year. Fusing multiple immunogenic antigens represents a promising approach to delivering an effective vaccine against leptospirosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a potential vaccine vector due to its adjuvant properties and safety. Two chimeric genes based on genic sequences of ligANI, ligBrep, lipL32, and lemA, were individually cloned into five BioBrick vectors with different promoters (pAN, Hsp60, 18 kDa, Ag85B and Ag85B plus signal sequence) for antigen expression in BCG. Groups of ten hamsters were vaccinated with recombinant BCG (rBCG) strains in two doses of 106 CFU and challenged with 5 × LD50 of L. interrogans serovar Copenhageni. All rBCG vaccines expressing chimera 1, based on antigens LipL32, LigANI, and LemA, under the control of any promoter, protected 80–100% of the hamsters from challenge (P < 0.05) and four of them also protected from renal carrier status; for chimera 2, based on LigANI and LigBrep antigens, the only vaccine that afforded survival rates statistically different from the control was the vaccine that incorporated the pAN promoter (60% of survival). A single vaccine dose was sufficient to induce significant IgG levels by all vaccine compositions evaluated; however, humoral response was not related to protection. These findings suggest that the combination of potential vaccine candidates in chimeric antigens and the use of BCG as a live vector are promising strategies by which it is possible to obtain an effective and sterilizing vaccine against leptospirosis.

Immune responses to killed reassorted influenza virus supplemented with natural adjuvants.

In this study, we investigated the immunomodulatory effects of a supplemented killed influenza virus (V) by Echinacea purpurea (E) and Nigella sativa (N) extracts and effect of changing the route of immunization from intramuscular (IM) to intraperitoneal (IP). At the 2nd-, 3rd- and 4th-week post-IM immunizations (WPIMI), the supplemented V with N (VN) induced the most significant IgM response unlike N alone. At the 2nd WPIMI, V or VN induced the highest significant IgG levels. At the 2nd-week post-IP immunization (WPIPI), E and VN induced the most significant IgG levels. Both at the 3rd and 4th WPIMI or WPIPI, various treatments induced significant increases in IgG. At the 4th WPIMI, E, V, and V with E (VE) induced significant increases in the CD4+ thymocytes while all IP treatments caused significant increase in their counts. V and VN induced the most significant IM induction of CD8+ thymocytes while their best IP stimulation was induced by N, VE, and VN. At the 4th WPIMI, various treatments caused significant increases in the mesenteric lymph node (MLN) CD4+, CD8+ counts. WPIPI with V or VE caused significant increases in both the CD4+- and CD8+ MLN cells, whereas VN significantly induced CD8+ MLN cells only. WPIPI with various treatments caused significant increases in the B-cell counts and the peak was obtained by VN.

Sequential colonization of periodontal pathogens in induction of periodontal disease and atherosclerosis in LDLRnull mice.

Periodontal disease (PD) and atherosclerotic vascular disease (ASVD) are both chronic inflammatory diseases with a polymicrobial etiology and have been epidemiologically associated. The purpose is to examine whether periodontal bacteria that infect the periodontium can also infect vascular tissues and enhance pre-existing early aortic atherosclerotic lesions in LDLRnull mice. Mice were orally infected with intermediate bacterial colonizer Fusobacterium nucleatum for the first 12 weeks followed by late bacterial colonizers (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) for the remaining 12 weeks mimicking the human oral microbiota ecological colonization. Genomic DNA from all four bacterial was detected in gingival plaque by PCR, consistently demonstrating infection of mouse gingival surfaces. Infected mice had significant levels of IgG and IgM antibodies, alveolar bone resorption, and showed apical migration of junctional epithelium revealing the induction of PD. These results support the ability of oral bacteria to cause PD in mice. Detection of bacterial genomic DNA in systemic organs indicates hematogenous dissemination from the gingival pockets. Bacterial infection did not alter serum lipid fractions or serum amyloid A levels and did not induce aortic atherosclerotic plaque. This is the first study examining the causal role of periodontal bacteria in induction of ASVD in LDLRnull mice.

Beneficial Effect of Exogenous PF4 on SRA for Detecting Pathogenic HIT Antibodies

Introduction: The laboratory diagnosis of HIT is difficult and often requires to perform an enzyme immunoassay combined with a functional assay, either serotonin release assay (SRA) or heparin-induced platelet aggregation (HIPA), for detecting IgG antibodies that activate platelets in the presence of heparin (anti-PF4/H antibodies). SRA is today considered as the gold standard for HIT diagnosis, but its performances could be improved as suggested by a recent clinical history and subsequent experiments.Case report: A 74 year-old man was hospitalized for a myeloma and treated with unfractionated heparin (UFH) for right atrium thrombosis. Thrombocytopenia and pulmonary embolism occurred the first day of heparin treatment and HIT was suspected. UFH was replaced by danaparoid sodium, and correction of platelet count was achieved within 3 days. Laboratory tests revealed the presence of significant levels of IgG to PF4/H (OD 1.2) with clearly positive HIPA. However, SRA was also performed to confirm the diagnosis of early onset HIT, but remained negative after testing platelets from two different donors. Unlike HIPA performed with PRP, SRA is done with washed platelets, and we thus hypothesized that PF4 concentration on cell surfaces might be too low for allowing heparin-dependent platelet activation when the patient's sample was tested. We thus performed a new SRA after adding exogenous PF4 (10 μg/ml) in the buffer and positive platelet activation pattern was obtained with a maximal release of 65% with 0.5 IU/ml UFH. This experience therefore prompted us to further evaluate whether addition of exogenous PF4 was beneficial or not on platelet activation induced by PF4/H antibodies when studied in SRA.Patients and methods: Plasma samples from 45 patients with suspected HIT and significant levels PF4-specific IgG were studied. SRA was systematically performed without and with exogenous PF4 (10 μg/ml). This "optimal" concentration of exogenous PF4 was defined after evaluating the effects of variable concentrations of human PF4 on platelet activation induced by 5B9 a recently developed HIT monoclonal antibody.Results: SRA was consistently negative when 5B9 was tested at concentrations lower than 5 μg/ml (1 or 2.5 μg/ml). In contrast, SRA was positive with these low 5B9 concentrations when 10 μg/ml of PF4 were present in the platelet buffer. When samples from suspected HIT were studied, conventional SRA (without PF4) was positive in 14 of 45 cases (Group SRA+), which all contained relatively high levels of PF4-specific IgG (median OD = 2.6). After addition of exogenous PF4, SRA was positive in 8 additional cases (Group SRAPF4+), with maximal release between 32% and 64% and a complete inhibition in the presence of 10 IU/ml UFH. Interestingly, levels of PF4-specific IgG were similar in this group and the 23 samples with whom SRA remained negative (median OD 1.6 vs. 1.3 respectively, p = 0.8). On the other hand, the pre-test probability of HIT (4Ts score) was always intermediate or high in 'SRA+' and 'SRAPF4+' groups, although it was low in more than 30% of patients with negative SRA.Discussion: Addition of PF4 in the platelet buffer might improve the detection of pathogenic HIT antibodies using SRA. Our results are in agreement with a recent study (I Nazi et al, Journal of Thrombosis and Haemostasis, 2015; 13: 1900-7), although they were obtained with a lower concentration of exogenous PF4, but which may be observed in vivo. DisclosuresNo relevant conflicts of interest to declare.

Norovirus (NoV) specific protective immune responses induced by recombinant P dimer vaccine are enhanced by the mucosal adjuvant FlaB.

BackgroundNoroviruses (NoVs) are a major cause of childhood gastroenteritis and foodborne diseases worldwide. Lack of appropriate animal models or cell-based culture systems makes the development and evaluation of NoV-specific vaccines a daunting task. VP1 is the major capsid protein of the NoVs that acts as a binding motif to human histo-blood group antigens (HBGAs) through its protruding 2 (P2) domain and can serve as a protective antigen candidate for vaccine development.MethodsRecombinantly produced NoV specific P domain (Pd) vaccine was inoculated into groups of mice either alone or in conjugation with mucosal adjuvant FlaB, the flagellar protein from Vibrio vulnificus. Antigen specific humoral and cell mediated immune responses were assessed by enzyme linked immunosorbent assay (ELISA) or fluorescent activated cell sorting (FACS). A comparative analysis of various routes of vaccination viz. intranasal, sublingual and subcutaneous, was also done.ResultsIn this study, we show that a recombinant Pd-vaccine administered through intranasal route induced a robust TH2-dependent humoral immune response and that the combination of vaccine with FlaB significantly enhanced the antibody response. Interestingly, FlaB induced a mixed TH1/TH2 type of immune response with a significant induction of IgG1 as well as IgG2a antibodies. FlaB also induced strong IgA responses in serum and feces. FlaB mediated antibody responses were toll like receptor 5 (TLR5) dependent, since the FlaB adjuvanticity was lost in TLR5−/− mice. Further, though the Pd-vaccine by itself failed to induce a cell mediated immune response, the Pd-FlaB combination stimulated a robust CD4+IFNγ+ and CD8+IFNγ+ T cell response in spleen and mesenteric lymph nodes. We also compared the adjuvant effects of FlaB with that of alum and complete Freund’s adjuvant (CFA). We found that subcutaneously inoculated FlaB induced more significant levels of IgG and IgA in both serum and feces compared to alum or CFA in respective samples.ConclusionWe validate the use of TLR5 agonist as a strong mucosal adjuvant that would facilitate the development of NoV specific vaccines for humans and veterinary use. This study also highlights the importance of route of immunization in inducing the appropriate immune responses in mucosal compartments.

GM-CSF and IL-4 Stimulate Antibody Responses in Humanized Mice by Promoting T, B, and Dendritic Cell Maturation

Engraftment of human hematopoietic stem cells into immunodeficient mice that lack T cells, B cells, and NK cells results in reconstitution of human blood lineage cells, especially B cells, in the recipient mice. However, these humanized mice do not make any significant level of IgG Ab in response to Ag stimulation. In this study, we show that in humanized mice, B cells are immature, and there is a complete deficiency of CD209(+) (DC-SIGN) human dendritic cells. These defects can be corrected by expression of human GM-CSF and IL-4 in humanized mice. As a result, these cytokine-treated humanized mice produced significant levels of Ag-specific IgG after immunization, including the production of neutralizing Abs specific for H5N1 avian influenza virus. A significant level of Ag-specific CD4 T cell response was also induced. Thus, we have identified defects in humanized mice and devised approaches to correct these defects such that the platform can be used for studying Ab responses and to generate novel human Abs against virulent pathogens and other clinically relevant targets.

Effective Treatment for Recurrent Pregnancy Loss

Understanding the etiology of recurrent pregnancy loss (RPL) has been increasing in importance in reproductive medicine, especially in societies with low birth rates. Since the cause of human miscarriage is often complicated and multifactorial, effective treatment for the successful maintenance of pregnancy requires the comprehensive clinical management of recurrent wastages. Immunology for the maintenance of pregnancy is a very attractive field; however, the role of immunology during human gestation cannot be confirmed in couples with a history of recurrent pregnancy loss unless other clinical causes of miscarriage are especially ruled out.Balanced chromosomal rearrangements have been found at an increased frequency in couples with recurrent pregnancy loss compared with the general population. The major anomalies are Robertosonian or reciprocal translocations, while most minor variants include pericentric inversion of chromosome 9. Both major and minor anomalies result in a spontaneous abortion rate of approximately 80-90%. Though the incidences of these chromosomal abnormalities vary in reported studies, a survey indicated an incidence of 9.8% (3.9% in male partners and 5.9% in female partners) among 1,639 couples with a history of recurrent pregnancy loss.Congenital uterine anomaly resulting in a suboptimal uterine environment may influence ovum implantation and early fetal development and may be a candidate in the etiology of reproductive wastage.A review on the relation between congenital uterine anomalies and the spontaneous abortion rate concluded that a precise diagnosis of uterine cavity deformity, especially arcuate uterus, is a key issue for determining the clinical prognosis during human gestation. After establishing diagnostic criteria for uterine cavity anomaly, 278 congenital uterine anomalies were detected in a series of 2,061 hysterosalpingographies (13.5%) performed in women with a history of recurrent pregnancy loss. Among these abnormalities, the incidences of spontaneous abortion were equally high among patients with arcuate uterus and those with partial septate uterus. A few studies on abortion mechanism in patients with congenital uterine anomalies have been reported. Histological findings using CD34- positive staining indicate the lack of a blood capillary system in both the uterine septum and the fundus muscle layer. The results of metroplastic surgery on congenital uterine anomaly are also controversial. Based on our fundamental findings of the maldistribution of uterine capillaries, we performed ideal metroplastic surgery in 173 women and significantly reduced the spontaneous abortion rate from a pre-operative values of 93.2% to a post-operative value of 12.5%.Anti-cardiolipin antibody was first described by Huge et al. as a pathogenic auto-antibody capable of causing recurrent pregnancy loss. Since then several other candidates for zwitterionic antiphospholipid antibodies to anionic phospholipids that are capable of inducing miscarriage such as phosphatidylethanolamine (aPE) and phosphatidylserine (aPS). In a multicenter study on antiphospholipid antibodies conducted at 15 insitutes and hospitals between 2000 and 2002, significant levels of IgG and IgM for aPE were detected among women with a history of recurrent pregnancy loss. By establishing an enzyme-linked immunosorbent assay system, we determined that the IgG and IgM levels for aPE were significantly higher than those for other phospholipids antibodies during the first trimester recurrent spontaneous abortions. Coagulation factor XII, prekallikrein and the high molecular weight molecule kininogen have been reported as plasma contact proteins in the intrinsic pathway of the blood coagulation system. Deficiencies of these proteins are not associated with clinical obstetrical intrauterine bleeding, suggesting that these proteins possess anticoagulant and profibrinolytic activities.(View PDF for the rest of the abstract.)

Antisperm antibodies detected by mixed agglutination reaction and immunobead test are not associated with chronic inflammation and infection of the seminal tract

The association between chronic inflammatory/infectious diseases of the male reproductive tract and the presence of antisperm antibodies (ASA) in semen is still controversial. We compared the results of the mixed agglutinin reaction (MAR) test and immunobead test for detecting ASA type IgG and IgA in 133 patients attending our special outpatient department for andrological infections and evaluated the differences in the detection rate of ASA. Patients were divided into three groups: a study group that included 79 patients with symptomatic nonacute inflammatory/infectious diseases of the seminal tract, a control group (n = 44) and a third group of men with a history of successful vasectomy reversal (n = 10). The two tests correlated in a statistically significant manner for the detection of IgG and IgA in all groups. The overall positive detection rate of clinical significant levels of IgG and IgA was 2.5% and 1.3% (respectively) in the patients with inflammation/infection of the seminal tract. No statistical significant difference in the detection rate of ASA levels between the inflammatory/infectious group and the controls was detected. The results of the MAR test and immunobead test have a statistical significant correlation and their results provide evidence that there is no association between inflammatory/infectious diseases of the male reproductive tract and the presence of ASA in semen.

A longitudinal investigation of Plasmodium falciparum malaria in children in northern India

A group comprising 27 young children (1-4 y of age) suffering from uncomplicated falciparum malaria were studied to characterize the isolates and to measure humoral immune responses during acute infection and after recovery. Finger prick blood from each individual was collected on d 1. After treatment with chloroquine, a further blood sample was collected from each child on d 7, 30, 90 and 180 for assay of antibody responses to P. falciparum antigens. Isolates from individual patients were incubated in vitro for demonstration of rosette formation, assay of plasmodial growth rate and analysis of Pfcrt gene polymorphism. Out of 27 isolates of P. falciparum, 20 showed formation of rosettes in vitro. The growth rate at 96 h varied widely among the isolates. In Pfcrt gene analysis at 76-codon site, 14 showed wild-type Lys 76, 7 showed mutant type Thr 76 and 6 had mixed type. 14 children, all with anaemia on d 7, showed a positive direct antiglobulin test (DAT). Sera positive by ELISA IgG on d 90 also showed parasite growth inhibitory activity in vitro. Significant levels of IgG, IgG1 and IgG3 subclass antibodies against MSP1 were detected in 14 sera collected on d 90. On d 180, there was a decline in IgG and its subtypes. These findings suggest that a variability in isolates may occur in one and the same seasonal area, making children prone to infection. As a consequence, they develop antibodies during recovery phase from an acute attack, which remain in circulation for a period of 4-5 months. After that, a decline in antibody level may again make them susceptible to the disease. Prevalence of different serotypes in a small area may suggest the complexity of malaria transmission.

Salmonella typhimurium grown in iron-rich media, inactivated with ferric chloride and adjuvanted with homologous bacterial DNA is potent and efficacious vaccine in mice

The present study describes our attempt to construct a novel vaccine formulation that affords full protection against murine typhoid under experimental conditions. Ferric chloride, 100 mM, as inactivating agent, bacterial growth under iron-rich conditions and homologous bacterial DNA as adjuvant were used for construction of the experimental Salmonella typhimurium vaccine. The vaccine inoculated twice at 2 weeks interval in Swiss albino mice elicited statistically significant IgG levels when compared with non-adjuvanted and other control groups. All the mice inoculated with the novel vaccine withstood challenge with 50 LD 50 dose of S. typhimurium strain St585. No significant safety problems were found in mice.

Fuchs Heterochromic Cyclitis and Ocular Toxocariasis

To report the association of Fuchs heterochromic cyclitis (FHC) and ocular toxocariasis in a young adult. Observational case report. A 26-year-old patient was referred for the management of a unilateral intermediate uveitis associated with a lower peripheral subretinal fibrotic lesion near the pars plana. Diagnosis of FHC was clinically confirmed. Laboratory examination was performed to exclude an infectious condition. LISA assay detected significant levels of IgG directed against Toxocara canis. Toxoplasmic serology was negative, excluding this differential diagnosis. Other examinations, including complete blood cell count, urinalysis, serum angiotensin-converting enzyme, lysosyme, chest CT scan, and syphilis serology were noncontributive. Previous studies have reported on the association of FHC and other parasitic conditions, such as toxoplasmosis but also on herpetic ocular infections. Serologic analysis for toxocariasis may be proposed in patients with FHC and retinal scars in the absence of toxoplasmosis.

A single dose sub-unit vaccine protects against pneumonic plague

In this study, the protection afforded against aerosolised Yersinia pestis by injection of a single dose of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine, prepared by admixing F1 antigen derived from a Y. pestis cell culture supernatant with recombinant V antigen derived from an E. coli cell lysate, fully protected an outbred strain of mouse against exposure to 10 6 CFU of virulent plague organisms (10 4 mouse lethal doses, MLD). In contrast, the whole cell vaccine provided only 16% protection against the same level of challenge. Furthermore, sub-unit vaccinees were able to clear the bacteria from their lungs post-challenge whereas bacteria were cultured from the lungs of a surviving KWC vaccinee post-challenge. In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable and the levels of F1-specific IgG in serum and in broncho-alveolar washings were significantly lower ( p<0.05) compared with sub-unit vaccinees. In sub-unit vaccinees, an IgG titre to the F1 and V antigens was detected in serum where it was significantly higher ( p<0.05) compared with broncho-alveolar washings suggesting that, at the time of challenge, protection is attributable mainly to the combined circulating IgG titre to the F1 and V sub-units. The enhanced protective efficacy of this sub-unit vaccine administered as a single dose compared with an existing vaccine has been demonstrated in an outbred animal model of pneumonic plague.

Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

Extraction and purification of active IgG from SSPE and MS brain

Immunoglobulin (Ig) G was purified from soluble and membrane fractions of postmortem subacute sclerosing panencephalitis (SSPE) brain, multiple sclerosis (MS) brain plaque-periplaque white matter, and normal human brain (NHB) white matter. After homogenization in 0.32 M sucrose and removal of cell debris and nuclei by low-speed centrifugation, soluble and crude membrane fractions were separated by ultracentrifugation. After removal of sucrose by dialysis, IgG was isolated from the soluble fraction by protein A affinity chromatography. IgG was obtained from the membrane fraction by elution at low pH and purification from the eluate by protein A chromatography. Whereas very little IgG was in NHB white matter, significant levels of IgG were recovered from both SSPE and MS brain. Both immunocytochemical staining of measles virus-infected cells in tissue culture and protein immunoblotting of virus-infected cell lysates showed that the IgG from SSPE brain contained activity specific for measles virus protein. The abundance, purity and functional activity of IgG extracted from SSPE and MS brain indicate that IgG extracted from the brain of humans with an inflammatory disease of unknown etiology can be used to identify its corresponding antigen.

Characterization of human immune responses to the cytosolic superoxide dismutase and glutathione S-transferase from Onchocerca volvulus.

In onchocerciasis patients and in O. volvulus-exposed individuals without signs of onchocericiasis, T- and B-cell responses to two recombinantly expressed O. volvulus enzymes were analysed and compared to responses to total protein extract of adult parasites. The cytosolic enzymes Cu/Zn superoxide dismutase 1 (OvSOD1) and glutathione S-transferase 2 (OvGST2) represent 2 detoxifying molecules which may play an important role in parasite defense against host-induced oxidative stress. The T-cell response to the two recombinant proteins was analysed by investigating the cytokine responses of peripheral blood mononuclear cells. Induction of IL-5 at the mRNA level and IL-5 and IL-10 at the protein level was demonstrated in patients with the generalized form of onchocerciasis and endemic normals without clinical manifestations. IFN-gamma was not found to be induced by either antigen. This pattern of lymphokine expression is indicative of a Th2-type response. Compared to patients with the generalized form, a higher level of cytokine induction was observed in the group of endemic normals. Low but significant IgG levels were observed against OvSOD1 in patients with onchocerciasis; higher antibody levels were found against OvGST2 in patients and endemic normals. The highest IgG levels were detected against the crude O. volvulus extract. These results indicate that the two recombinant O. volvulus proteins induce moderate T and B cell responses.