Blood-stage immunity to Plasmodium chabaudi malaria following chemoprophylaxis and sporozoite immunization.

Protection against malaria in humans can be achieved by repeated exposure to infected mosquito bites during prophylactic chloroquine treatment (chemoprophylaxis and sporozoites (CPS)). We established a new mouse model of CPS immunization to investigate the stage and strain-specificity of malaria immunity. Immunization with Plasmodium chabaudi by mosquito bite under chloroquine cover does not generate pre-erythrocytic immunity, which is acquired only after immunization with high sporozoite doses. Instead, CPS immunization by bite elicits long-lived protection against blood-stage parasites. Blood-stage immunity is effective against a virulent, genetically distinct strain of P. chabaudi. Importantly, if exposure to blood-stage parasitemia is extended, blood-stage parasites induce cross-stage immunity targeting pre-erythrocytic stages. We therefore show that CPS immunization can induce robust, long-lived heterologous blood-stage immunity, in addition to protection against pre-erythrocytic parasites following high dose sporozoite immunization. Cross-stage immunity elicited by blood-stage parasites may further enhance efficacy of this immunization regimen.

Assessment of chloroquine as a modulator of immune activation to improve CD4 recovery in immune nonresponding HIV-infected patients receiving antiretroviral therapy.

OBJECTIVES: Chloroquine (CQ), an anti-inflammatory drug, inhibits Toll-like receptor (TLR) signalling in plasmacytoid dendritic cells (pDCs) and may be beneficial for HIV-infected patients in whom immune activation persists despite effective antiretroviral therapy (ART). The effect of CQ on CD4 T-cell recovery and immune activation in immune nonresponding patients receiving successful ART was therefore studied. METHODS: Nineteen adults on ART with CD4 counts ≤ 350 cells/µL and undetectable viral load (VL) orally received CQ at 250 mg/day for 24 weeks. Side effects, CD4 and CD8 T-cell counts, VL, T-cell activation, pDC proportion and plasma inflammatory markers were assessed at baseline, at 24 weeks, and at 12 weeks after CQ discontinuation (clinicaltrial.org registration #NCT02004314). RESULTS: CQ was well tolerated and all patients maintained an undetectable VL. The absolute CD4 and CD8 T-cell counts and their percentages, the pDC proportion, T-cell activation, D-dimer and C-reactive protein (CRP) plasma levels and the kynurenine/tryptophan ratio did not change with CQ treatment. Among nine cytokines/chemokines measured, only levels of interferon (IFN)-α2 were significantly increased by CQ treatment. CONCLUSIONS: CQ was well tolerated in patients with low CD4 T-cell counts despite long-term effective ART; however, 24 weeks of CQ treatment did not improved CD4 T-cell recovery, lymphoid and myeloid immune activation or inflammatory markers.

A nanovector with complete discrimination for targeted delivery to Plasmodium falciparum-infected versus non-infected red blood cells in vitro.

Current administration methods of antimalarial drugs deliver the free compound in the blood stream, where it can be unspecifically taken up by all cells, and not only by Plasmodium-infected red blood cells (pRBCs). Nanosized carriers have been receiving special attention with the aim of minimizing the side effects of malaria therapy by increasing drug bioavailability and selectivity. Liposome encapsulation has been assayed for the delivery of compounds against murine malaria, but there is a lack of cellular studies on the performance of targeted liposomes in specific cell recognition and on the efficacy of cargo delivery, and very little data on liposome-driven antimalarial drug targeting to human-infecting parasites. We have used fluorescence microscopy to assess in vitro the efficiency of liposomal nanocarriers for the targeted delivery of their contents to pRBCs. 200-nm liposomes loaded with quantum dots were covalently functionalized with oriented, specific half-antibodies against P. falciparum late form-infected pRBCs. In less than 90min, liposomes dock to pRBC plasma membranes and release their cargo to the cell. 100.0% of late form-containing pRBCs and 0.0% of non-infected RBCs in P. falciparum cultures are recognized and permeated by the content of targeted immunoliposomes. Liposomes not functionalized with antibodies are also specifically directed to pRBCs, although with less affinity than immunoliposomes. In preliminary assays, the antimalarial drug chloroquine at a concentration of 2nM, ≥10 times below its IC(50) in solution, cleared 26.7±1.8% of pRBCs when delivered inside targeted immunoliposomes.

Empowering malaria vaccination by drug administration.

Although significant progress has been made in clinical development, a protective malaria vaccine remains elusive. Here we review some of the immune subversive mechanisms used by the Plasmodium malaria parasite and propose a potentially effective strategy to achieve complete protection that may serve as a blue print for clinical usage. The premise is to modulate the immune response with drugs that neutralize suppressive functions and potentiate protective responses. Chloroquine may be a first attractive candidate facilitating protective cellular immune responses by improving cross-presentation and reducing suppressive regulatory T cell responses.

Chloroquine has not disappeared.

Chloroquine (CHQ), an antimalarial, is also used as an anti-inflammatory drug for systemic lupus erythematosus and rheumatoid arthritis (RA). Hydroxychloroquine (HCQ) reduces the frequency of organ involvement and disease flares, and relieves skin and joint symptoms. CHQ reduces the immunologically-mediated inflammation of the joints. HCQ and combination therapies have a significant benefit on synovitis, pain and physical disability on RA. We advocate the investment of resistance Plasmodium prevalence determinations in countries beset by malaria, and to match thereafter the quantity of persons administered CHQ. Follow-up investigations are essential to diagnose and prevent visual damage.

Immunohistochemical demonstration of the distribution of chloroquine (CQ) and its metabolites in CQ-poisoned mice.

Chloroquine (CQ) distribution in tissues of acutely poisoned mice was demonstrated by immunohistochemistry using anti-CQ polyclonal antibodies (PAC). PAC recognized 4-amino-7-chloro-quinoline structure and sufficiently reacted with CQ and CQ's metabolite bisdesethyl-chloroquine. In the brain, CQ and its metabolites (CQs) localized in the region of the choroids plexus, indicating an important role in the blood-cerebrospinal barrier system. In the heart, most regions showed diffused positive staining, and relatively strong reaction was observed in Purkinje cells, indicating an important role in acute CQ toxicity. In the lungs, CQs were observed in the bronchial epithelium, type II pneumocytes, and on the surface of alveolar walls. It was suggested that CQs were excreted to the alveolar wall with surfactant phospholipids, which are produced by type II pneumocytes. In the liver, CQs were concentrated in the centrolobular area rather than in the periportal area, in agreement with CQ's metabolic pathway. In the kidneys, tubular cells were strongly stained compared to glomerular capsules, and the distal part of renal tubules was better stained than the proximal tubules. These findings suggested that CQs were predominantly excreted or reabsorbed through the distal tubules and the collecting duct. Distribution of CQs in tissues presented here were mostly consistent with the physico-chemical properties of CQ and its metabolites. However, the elucidation of CQs' localization in Purkinje cells remains open. Further experimental studies at the level of microorganella will be needed to clarify the present result.

A mannose-receptor is possibly involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) leucocytes.

In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes. Leucocytes were preincubated for 30min with different concentrations of sugar (alpha-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays. The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry. The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent. More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked. Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed. The results demonstrated that the phagocytosis of yeast was completely blocked by this substance. The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes.

Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs.

An inflammatory response is invariably associated with administration of gene transfer complexes composed of cationic lipids and plasmid DNA (pDNA). In the lung, an influx of neutrophils and elevated levels of several proinflammatory cytokines such as TNF-alpha, IFN-gamma, IL-6, and IL-12 characterize this dose-dependent response. The induction of these cytokines was shown previously to be due in part to the presence of unmethylated CpG dinucleotides in the bacterially derived pDNA. We have eliminated 270 of 526 CpG dinucleotides in a reporter plasmid (pCFA-CAT) and tested the inflammatory response to cationic lipid:pDNA complexes containing the modified vector (pGZA-CAT) after intravenous (i.v.) or intranasal (i.n.) delivery into BALB/c mice. Compared to the unmodified vector, the CpG-reduced pGZA-CAT was found to be significantly less immunostimulatory, as the levels of IL-12, IFN-gamma, and IL-6 in the serum 24 h after i.v. delivery were reduced by 40 to 75%. Similar reductions in cytokine levels were also observed in the bronchoalveolar lavage fluids (BALF) after i.n. administration, while the levels of reporter gene expression were not affected by the modifications. We have also investigated known inhibitors of the CpG signaling pathways in order to decrease the inflammatory response. Two such inhibitors, chloroquine and quinacrine, greatly reduced the induction of IL-12 from mouse spleen cells in vitro and inhibited cytokine production in the lung by approximately 50% without affecting gene expression. These results illustrate that use of a less immunostimulatory pDNA vector or inhibitors of CpG immunostimulation can reduce significantly the toxicity associated with cationic lipid:pDNA complexes thereby increasing the therapeutic index of this synthetic gene transfer vector.

Stereospecificity of antibody: quinine, the optical isomer of quinidine and anti-malarial drug chloroquine do not cross-react with quinidine immunoassays.

Quinine is an optical isomer of quinidine. Both quinine and chloroquine (an aminoquinoline derivative) are used in treating malaria. The authors studied cross-reactivity of quinine and chloroquine with the quinidine immunoassays using the TDx and AxSYM analyzers (Abbott Laboratories, Abbott Park, IL). The authors observed no cross-reactivity of chloroquine with quinidine immunoassays (TDx and AXSYM) even when drug-free serum was supplemented with 1000 microg/mL chloriquine. The authors observed no cross-reactivity of quinine up to a concentration of 250 microg/mL. At higher concentrations, the authors observed a small cross-reactivity. The cross-reactivity of a substance should be studied in the presence of the primary analyte. When serum pools prepared from patients receiving quinidine were supplemented with various concentrations of quinine or chloroquine, the authors observed statistically significant declines in quinidine concentrations with higher concentrations of both quinine and chloroquine. The authors observed significant cross-reactivity of L-amphetamine with the amphetamine immunoassay also marketed by Abbott Laboratories and run on the AxSYM analyzer. The authors conclude that although the antibody used in the quinidine assay is stereospecific, the antibody used in the amphetamine assay by the same manufacturer is not stereospecific.

Antibodies reacting with chloroquine in human sera.

Presence of antibodies in human sera reacting with chloroquine (CQ) was demonstrated from a malaria endemic belt. The study groups consisted of 53 healthy volunteers, receiving regular chloroquine prophylaxis [2 tablets of CQ (150 mg)/wk, single dose], 105 individuals with a history of frequent CQ intake for any febrile attack and 95 individuals from a non-endemic area who had neither a history of malaria nor of previous consumption of CQ. Sixty six per cent individuals taking regular prophylactic chloroquine and about ten per cent of those with a history of taking frequent chloroquine due to febrile illness in the malaria endemic area showed antibodies reacting to chloroquine. However, antichloroquine antibodies were either insignificant or nondetectable in serum samples collected from persons without any history of chloroquine intake. The results indicate a possible interaction of CQ in vivo.

Human immunodeficiency virus status and delayed-type hypersensitivity skin testing in Ugandan children.

BACKGROUND: In previous studies, delayed-type hypersensitivity (DTH) skin testing has been shown to be affected by several factors including nutritional status, intercurrent infection, host immune status, and previous exposure to the antigen being used. OBJECTIVE: To determine the effect of human immunodeficiency virus type 1 (HIV-1) status on DTH skin testing in a cohort of HIV-1-infected and noninfected Ugandan children followed prospectively from birth. DESIGN: Nested case-control study. SETTING: Primary care clinic serving study participants at Mulago Hospital, Makerere University, Kampala, Uganda. PARTICIPANTS: Thirty HIV-1-infected children and 30 age-matched, HIV-1-noninfected children. METHODS: After completion of history and physical, each child underwent Mantoux skin testing with both Candida and purified protein derivative (PPD). Results of skin testing were read in 48 to 72 hours. Complete chart reviews were performed on all children. CD4 lymphocyte counts were obtained on all HIV-1-infected children at the time the skin testing was read. RESULTS: The average age of participants was 67 months (range, 51-92 months). HIV-1-infected children (mean CD4 lymphocyte count, 1069 mL-1; range, 86-3378 mL-1), compared with noninfected, age-matched peers, developed significantly smaller PPD reaction size (mean, 1.18 mm +/- 4.3 vs 3.6 mm +/- 7.6, respectively). Candida responses were not different between the two groups of children. Among HIV-1-infected children, there was a larger Candida reaction size in children who had recently received chloroquine treatment. There was no significant correlation between Candida reactivity and PPD reactivity, progressive HIV-1 disease, or CD4 lymphocyte count. The six children diagnosed clinically with active tuberculosis had lower absolute CD4 lymphocyte counts than children without tuberculosis. Lack of reaction to PPD was associated with lower CD4 lymphocyte counts and progressive HIV-1 disease. CONCLUSIONS: In HIV-1-infected Ugandan children, DTH skin testing was influenced by the choice of antigen selected, HIV-1 infection, and recent treatment with chloroquine. Based on these findings, we believe that further prospective, longitudinal investigation into the role of chloroquine in HIV-1-infected children is needed. We emphasize the limitations of DTH skin testing in HIV-infected children as an adjunct in the diagnosis of active tuberculosis.

Malaria chemosuppression in pregnancy. III. Its effects on the maternal malaria immunity.

The malaria immune status of pregnant women participating in a malaria prophylaxis study was assessed using their sera reactivity to the R32tet32 antigen. Supervised prophylaxis started early in pregnancy till delivery. Women randomly received either chloroquine once weekly (CQ), or proguanil daily (PROG), or a combination of the two drugs (CQ + PROG). Blood was collected at enrollment, then after 8, 16, and 24 weeks of prophylaxis. Of the 312 women who received prophylaxis for more than 10 consecutive weeks before delivery, anti-sporozoite antibodies were assayed in 232 at enrollment, 258 after 8 weeks, and 254 after 16 weeks. Titres at enrollment were comparable by parity and the residential area. Antibodies in women of the PROG group who were parasitaemic before the assessments decreased with the increasing number of breakthrough and clinical episodes. The converse was true for antibodies in the CQ and CQ + PROG groups. Group differences in the parasite densities would explain this. Parity and placental malaria did not influence titres significantly. Overall, antibodies of the CQ + PROG group decrease significantly with time, possibly due to its long period of aparasitaemia. This suggested interference with the immune responsiveness of the women. PROG, which was equally efficacious, offers better prophylaxis.

The pfmdr gene homologues of Plasmodium falciparum.

Chloroquine resistance in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumour cells which is mediated by P-glycoprotein. P. falciparum has two mdr-like genes (pfmdr 1 and pfmdr 2) and pfmdr 1 has been linked to the chloroquine resistance phenotype. We show that pfmdr 1 encodes a protein of 160,000 Daltons that is expressed at higher levels in a chloroquine resistant cloned isolate. The pfmdr 2 gene is located on chromosome 14 and it is in equal copy number in chloroquine resistant and sensitive isolates. Therefore amplification of pfmdr 2 is not linked to chloroquine resistance. This is in contrast to the pfmdr 1 gene which has been shown to be amplified in some chloroquine resistant isolates.

The effect of short-term malaria chemoprophylaxis on the immune response of semi-immune adult volunteers.

In 17 semi-immune adult volunteers, chemoprophylaxis with 300 mg chloroquine base weekly for six months was found to be effective in suppressing malaria. However, following 3 months of chemoprophylaxis, a significant reduction of IFA titres was seen lasting up to 2 months after chloroquine withdrawal. There was resurgence of malaria in the post-intervention phase. 2 months after drug withdrawal, serum concentrations of IgG and factor B were significantly reduced. 3 months after initiation of chloroquine prophylaxis, a temporary but significant decrease of IgG and IgM serum concentrations was found with a corresponding decline in the number of B-lymphocytes and regulatory T-cells. These returned to normal at 6 months of chemoprophylaxis. Our findings suggest that short-term malaria chemoprophylaxis may significantly interfere with humoral and cell-mediated immunity in areas of intensive P. falciparum transmission.

A specific ELISA method for determining chloroquine in urine or dried blood spots.

Reported is an enzyme-linked immunosorbent assay (ELISA) that has been optimized and validated for the determination of chloroquine in urine or dried blood spots. The assay employs antisera raised in sheep to a chloroquine derivative conjugated to keyhole limpet haemocyanin and chloroquine conjugated to porcine thyroglobulin adsorbed onto the wells of a microtitration plate. The competitive binding of the antiserum to the wells was monitored using an alkaline-phosphatase-conjugated second antibody and a specific substrate. The assay exhibits no cross-reactivity with known chloroquine metabolites, other antimalarials, and commonly used drugs. The method was used to determine chloroquine in dried blood spot extracts and urine from a patient who was receiving a prescribed prophylactic chloroquine regimen. The drug was detected in the urine for 17 weeks and in the dried blood spots for 4 weeks after termination of the therapy.

Monoclonal antibodies to lipophilic and short-sized haptens: application to the 4-amino-quinoline antimalarial drugs.

Monoclonal antibodies recognizing the 4-amino-7-chloro-quinoline (ACQ) structure, which represents the backbone of the 4-amino-quinoline antimalarial drugs, were obtained in mice, after injection of ACQ coupled to hemocyanin via the glutaraldehyde method. The resulting antibodies show a definite specificity to this hapten, but react better with compounds substituted on the exocyclic amino group in 4. It is postulated that the quinoline ring is not sufficient for the reaction with the antibodies, and that an enlarged structure, which is given by the bridge used to link hapten and carrier, entails an important increase (1000-fold) in the apparent affinity. The striking similarities between this bridge and the lateral chains of the antimalarial drugs are accountable for this enhanced recognition. This result allows us to indicate that in some instances, the bridge-structure of the immunogen should be positively involved in the epitope. This observation may become useful in the conception of immunogens, aiming to obtain antibodies directed against some lipophilic and small-sized haptens.

Large, but not small, antigens require time- and temperature-dependent processing in accessory cells before they can be recognized by T cells.

We have studied if antigens of different size and structure all require processing in antigen-presenting cells of guinea-pigs before they can be recognized by T cells. The method of mild paraformaldehyde fixation was used to stop antigen-processing in the antigen-presenting cells. As a measure of antigen presentation we used the proliferative response of appropriately primed T cells during a co-culture with the paraformaldehyde-fixed and antigen-exposed presenting cells. We demonstrate that the large synthetic polypeptide antigen, dinitrophenyl-poly-L-lysine, requires processing. After an initial time-lag of 30 min this antigen is fully processed within 2 to 4 of culture at 37 degrees C. In contrast, the immunogenic heptapeptide, angiotensin III, can be presented by pre-fixed accessory cells, viz. without any prior processing. Antigen processing was found to be temperature-dependent and consequently energy-requiring. Processing is strongly inhibited by the lysosomotrophic drug, chloroquine, suggesting a lysosomal involvement in antigen processing. The existence of a minor, non-lysosomal pathway is suggested, since small amounts of antigen were processed even at 10 degrees C, at which temperature no transport to and from the lysosomes can occur.

Effect of selected immunoregulatory agents on low-grade contact sensitivity.

The effect of selected compounds with known immunoregulatory activity was examined in a 45-h sensitization period oxazolone contact-sensitivity reaction. Oxazolone sensitivity was induced by applying 0.1 ml of 5% oxazolone in absolute ethanol to the shaved abdomen of C57Bl/6 mice on day 0. Challenge with oxazolone followed 45 h later and was accomplished by painting a 5% solution of oxazolone in absolute ethanol on the left hindpaw. The response at 24 h was determined plethysmographically. Histamine (0.062-1.0 mg/kg, subcutaneously, twice a day), concanavalin A (0.31-5.0 mg/kg intravenously), penicillamine (6.25-25 mg/kg, subcutaneously), chloroquine (6.25-25 mg/kg, subcutaneously), and thymosin fraction 5 (0.125-1.25 mg/kg subcutaneously) all stimulated the oxazolone reaction when administered on day 0. These data suggest that the low-grade oxazolone response may be a useful assay to detect immunostimulatory activity.