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The current therapies against cancer showed limited success. Nanotechnology is a promising strategy for cancer tracking, diagnosis, and therapy. The hybrid nanotechnology assembled several materials in a multimodal system to develop multifunctional approaches to cancer treatment. The quantum dot and polymer are some of these hybrid nanoparticle platforms. The quantum dot hybrid system possesses photonic and magnetic properties, allowing photothermal therapy and live multimodal imaging of cancer. These quantum dots were used to convey medicines to cancer cells. Hybrid polymer nanoparticles were utilized for the systemic delivery of small interfering RNA to malignant tumors and metastasis. They allowed non-invasive imaging to track in real-time the biodistribution of small interfering RNA in the whole body. They offer an opportunity to treat cancers by specifically silencing target genes. This review highlights the major nanotechnology approaches to effectively treat cancer and metastasis.

International audience; A new method for the detection by flow cytometry of anti-double-stranded DNA antibodies and of circulating immune complexes (IC) containing endogenous DNA (IC-eDNA) is described. From each serum sample, two samples were taken, one was used to detect IC-eDNA. The other to detect anti-DNA antibodies was incubated with calf thymus DNA. ICs were isolated by polyethylene glycol precipitation or by cryoprecipitation, after which immunoglobulins were labeled with FITC-conjugated anti-human globulin. Serum samples from 63 systemic lupus erythematosus (SLE) patients, 32 incomplete lupus, and 87 control patients were tested. Detection of anti-dsDNA antibodies by flow cytometry had a diagnostic sensitivity and specificity almost comparable to routine tests, the fluorescent enzyme immunoassay EliA™-dsDNA test, and the ultrasensitive Crithidia luciliae indirect immunofluorescence test. In 21 (33%) out of 63 SLE serum samples, IC-eDNA was detected. In these samples, free anti-dsDNA antibodies were hardly detectable or undetectable by flow cytometry or by routine tests. When anti-DNA antibodies are neutralized by endogenous DNA and can no longer be detected by routine tests, the serologic diagnosis and the follow-up of relapses in patients with SLE is compromised. To overcome this obstacle, we propose an accessible solution: the detection of circulating IC-eDNA by flow cytometry.

Phospholipids (PLs) are the target for the clearance of apoptotic cells, and for the innate defense against malignant cancers or microbial infections. Several types of receptors for PLs were described on T lymphocytes, but not yet on B lymphocytes. To detect PLs binding lymphocytes two assays were used: a- FITC labelled liposomes, b- Petri dish coated with different PLs. The phenotype of lymphocytes was determined by FACS, using anti-CD mAb and anti-Ig polyclonal antibodies. Human lymphocytes bind PLs, the binding ratio decreases in the order phosphatidylserine (PS), cardiolipin (CL), phosphatidylethanolamine, and phosphatidylcholine. In healthy blood donors (n=17), 29% of B lymphocytes, 17% of NK cells, 9% of TCD4 + , and 4% of TCD8 + bind PS. Among B cells, the binding ratio varies with the subset, 49% for naive cells (sIgM + sIgD + ), 42% for sIgA + , and 15% for sIgG + B cells. PL binding was not affected by the CD5 phenotype of B lymphocytes, nor by the HIV status of the donors (n=16) or the presence in the sera of IgG anti- CL antibodies. Pretreatment of lymphocytes with proteases inhibited the binding of PL, suggesting that binding is due to surface proteins. For each type of lymphocytes the various kinds of surface proteins which bind lipids, pattern recognition receptors, surface immunoglobulins, the lipid antigen presenting molecules CD1, and the CD1-restricted lipid antigen reactive TCR of NKT cells, are discussed.

Summary Background To confirm allergy to b-lactam (BL), a basophil activation test in flow cytometry based on CD63 up-regulation was described. CD203c is a more recent basophil activation marker and up to day there is no consensus about which marker is the more sensitive one. CD203c has not yet been evaluated in the diagnosis of BL allergy. Objective The aim of the study was to compare the reliability of CD203c to CD63 for the diagnosis of amoxicillin (AX) allergy, which is nowadays the most frequent BL allergy. Methods Twenty-seven patients with an immediate positive skin test (ST) to AX, 20 had had anaphylaxis with AX and 7 had urticaria and/or angioedema, were compared with 14 controls with no allergy to BL and to six patients with delayed positive ST to AX. Results In the anaphylaxis group, AX induced up-regulation of CD203c in the basophils of 12 patients out of 20 (60%) and of CD63 in four patients (20%) (Po0.02). Two patients out of seven with urticaria or angioedema had a positive result with CD203c and CD63. In patients who had anaphylaxis, ampicillin (AMP) induced CD203c up-regulation in eight out of 12 (67%) patients tested, and CD63 up-regulation in 4 out of 12 (33%) (all patients who had anaphylaxis could not be tested with AMP). False-positive results were observed with CD203c as well as CD63, and for 10 patients indeed this was confirmed by a negative drug provocation test. The origin of conflicting results between CD63 and CD203c might be at least the targeting of basophils based on anti-IgE labelling. Among IgE 1 gated cells, by means of CD33, a marker of monocytes, a contamination up to 50% by monocytes was detected. In contrast to CD63, CD203c is an activation marker specific of basophils with a basal low-level expression in resting basophils. Thus, IgE and CD203c double targeting of basophils avoids the contamination by monocytes. Conclusion CD203c seems to be a more sensitive activation marker of basophils than CD63 for the diagnosis of amoxicillin allergy.

Resume La recherche et la quantification des anticorps anti-DNA natif (dsDNA) sont utiles pour le diagnostic et le suivi du lupus erythemateux dissemine. Differents tests sont utilises, bases sur des methodologies variees qui peuvent influencer les resultats. Cette etude a evalue la specificite et la sensibilite du test dsDNA Liaison ® Diasorin pour la detection des anticorps anti-dsDNA. Nous avons compare les resultats au test ELIA ® dsDNA Pharmacia utilise en routine dans notre laboratoire. Les serums provenant de 153 patients avec un lupus erythemateux dissemine et de 78 temoins ont ete etudies. Les temoins comprennent 54 maladies auto-immunes et 24 patients sans marqueurs d'auto-immunite. La sensibilite du test est de 91,5 % et sa specificite superieure a 99 %. La correlation avec le test ELIA ® dsDNA Pharmacia est de 0,68. Le test automatise Diasorin Liaison ® dsDNA a montre ses performances pour la detection et la quantification des anticorps anti-dsDNA chez les patients lupiques.

Background: Anaphylactic reactions during anesthesia are mainly the result of muscle-relaxant (MR) drugs. Skin tests, serologic detection of specific IgE, and in vitro leukocyte histamine release are used to investigate MR allergy. Objective: We describe a new assay that is based on the detection by flow cytometry of the altered expression of plasma membrane molecules of MR-activated basophils. Methods: For this assay, which we have named the BASIC assay, basophils are incubated in vitro with MR, after which they are fixed and then triple labeled with fluorescein-conjugated anti-CD63, tandem dye R-phycoerythrin-cyanin 5.1 conjugated anti-CD45, and R-phycoerythrin conjugated anti-IgE. The resulting B asophils' A ltered S urface I mmunofluorescence is detected by flow C ytometry (BASIC). Results: Forty-one patients who had an allergic reaction during general anesthesia and 23 control subjects without such a history were studied. All included subjects' basophils were tested in the BASIC assay with at least 4 MR: suxamethonium, gallamine, vecuronium, and pancuronium. After reaction of the basophils of the MR-allergic patients with MRs, increased surface expression of CD63 and CD45 and decreased expression of IgE were detected. Increased expression of CD63 was observed most frequently and it was stronger than the alteration of the 2 other markers. Cross-reactivity between MRs commonly occurred. MRs diluted 10 –1 activate the basophils of the control subjects, suggesting that at relatively high concentrations MRs are also nonspecific basophil activators. Conclusion: In the diagnosis of MR allergy, the BASIC assay has a good specificity but a low sensitivity, and it correlates strongly with skin test results. It is currently appraised for the diagnosis of anaphylactic reaction induced by other classes of drugs. (J Allergy Clin Immunol 1999;104:411-8.)

Background. An in vitro basophil activation test, based on the detection of CD63 upregulation induced by NSAIDs, has been described. Its clinical significance remains controversial. Objectives. In patients with a history of nonallergic NSAID hypersensitivity, stratified according to the severity of the symptoms, to assess with NSAIDs the predictive value of basophil (BAT) and monocyte (MAT) activation tests. Patients/Methods. Sixty patients who had NSAIDs-induced or exacerbated urticaria/angiooedema and 20 controls was included. After incubation with NSAIDs or acetaminophen, leukocytes were analysed for CD63 upregulation. Results. With aspirin, the sensitivity (37%) and specificity (90%) of BAT agree with already published results. In contrast, when patients had had cutaneous and visceral reactions, the frequency of positive BAT 14/22 (64%, P<0.001) or MAT 10/22 (46%, P<0.01) were increased. Conclusions. Positive tests were more frequent among patients having a severe hypersensitivity contrasting with the other patients who had results similar to controls.

The specificity and sensitivity of anti-SSA (Ro) antibody detection by double immunodiffusion, ELISA, western blot and a new method named CAPE blot were studied. Using 79 normal sera, 61 sera positive for anti-SSA (Ro) antibodies (double immunodiffusion), and 39 sera without anti-SSA (Ro) antibodies but containing anti-Sm, anti-RNP, anti-SSB (La), anti-Scl 70 and anti-PCNA antibodies, we compared ELISA, western blot and CAPE blot with the immunodiffusion assay. The sensitivities of the three methods were 100, 62 and 100%, respectively. Sera from 196 patients with systemic lupus erythematosus (SLE) were tested. The prevalence of anti-SSA (Ro) antibodies in this group was scored as 26% by double immunodiffusion, 47% by ELISA, 25% by western blot and 38% by CAPE blot.

Resume Les anticorps anti-SSB ont ete doses au moyen de deux techniques immuno-enzymatiques de type ELISA. La difference entre celles-ci reside dans la preparation de l'extrait antigenique SSB. La premiere, mise au point dans notre laboratoire, utilise un antigene SSB purifie a partir de thymus de veau, la seconde un antigene clone par genie genetique. Nous avons realise une etude analytique de reproductibilite intra-essai, inter-essai et limite de detection de notre technique ELISA et affine son evaluation par une etude clinique portant sur 203 sujets (55 presentant un syndrome de Sjogren, 47 un lupus, 17 une polyarthrite rhumatoide, 13 une sclerodermie, 10 une polymyosite et 61 temoins). Les resultats obtenus ont ete compares a la methode de reference d'Ouchterlony. Les deux methodes ELISA sont concordantes (coefficient de correlation global de 0,96) malgre la difference de preparation de l'extrait antigenique. L'immuno-enzymologie est beaucoup plus sensible que la technique d'Ouchterlony puisque, pour une population de Sjogren, nous obtenons une sensibilite de 0,69 alors qu'elle n'est que de 0,51 pour l'immunodiffusion. Cette augmentation de sensibilite se retrouve pour la population de lupus, ou l'on obtient les chiffres de 0,42 pour la technique ELISA et 0,25 pour l'immunodiffusion double d'Ouchterlony.

Recently antibodies to hepatitis C virus were detected in sera of chronic active hepatitis patients, with anti-smooth muscle autoantibodies or with anti-liver/kidney microsomal type 1 autoantibodies. As the latter were used to differentiate autoimmune chronic active hepatitis from chronic non-A, non-B virus hepatitis, it was mainly important to discover if autoantibodies were associated with chronic hepatitis C virus infection. The sera of 272 chronic hepatitis C patients were screened by indirect immunofluorescence for non-organ specific autoantibodies. Antinuclear anti-bodies and anti-smooth muscle autoantibodies were more frequent in chronic hepatitis C patients than in blood donors ( n = 100). Anti-liver/kidney microsomal type 1 autoantibodies were not detected in the sera of the blood donors, in the 74 hepatitis B patients or in the 30 alcoholic hepatitis or cirrhotic patients' sera tested as controls. They were detected in 14 chronic hepatitis C patients. These antibodies were compared in immunodiffusion to anti-liver/kidney microsomal type 1 autoantibodies sera obtained from type-2 autoimmune chronic active hepatitis patients and an identity reaction was observed. Chronic hepatitis C patients without or with anti-liver/kidney microsomal type 1 autoantibodies, did not differ in age, sex ratio, transaminases and gammaglobulin level, risk factors for hepatitis C virus infection, association with other autoimmune diseases. These patients differed significantly from type-2 autoimmune chronic active hepatitis patients. We conclude that: (i) in some chronic hepatitis C patients the pattern and the titer of autoantibodies may create confusion with an autoimmune chronic active hepatitis; (ii) There is no serological evidence for a hepatitis C virus infection in true type-2 autoimmune chronic active hepatitis.

A 66-year-old patient, undergoing heart surgery, developed an anaphylactic reaction following the first administration of a test-dose of aprotinin. Skin tests were performed six months later. Prick-tests with 10(-2) and 10(-1) aprotinin dilutions were negative but intradermal reaction with a 10(-3) dilution was clearly positive. The level of aprotinin specific IgE was high, both in the serum obtained before surgery and in the one sampled on the day of the testing. As no reaction was observed during the tests, skin-testing and specific IgE appear to be an interesting alternative to a potentially dangerous test-dose, but they both need further evaluation.

Immunodeficiency related to HIV may increase the incidence of EBV-associated lymphomas, by altering EBV-specific immune control and consequently favoring EBV reactivation. The aim of the present study was to assess the relationship between the decrease of EBV-specific cellular immunity and the increase of EBV reactivation in a prospective cohort of 72 unselected HIV-infected individuals. EBV-specific immunity was evaluated by a highly sensitive IFN-gamma ELISPOT assay using 22 peptides mimicking latent and lytic antigens, and circulating mononuclear (PBMC) EBV DNA load was quantified by real-time quantitative PCR. The mean circulating cell-associated EBV DNA load was higher in HIV-infected patients (639 copies/10(6) PBMC) than in healthy controls (21, n = 14) ( P = 0.005) and was higher in patients with CD4(+) T-cell count below 350/microL than that in patients harboring higher CD4(+) T-cell count (1112 vs. 389, P = 0.003). The mean intensity of EBV-specific cellular responses was lower in HIV-infected patients than in controls ( P = 0.001), even in patients with CD4(+) T-cell count above 350/-microL ( P = 0.007). The number of EBV peptides recognized was lower in HIV-infected patients than in controls (frequency: 0.44 vs. 0.67; P = 0.02), indicating reduced polyclonality in HIV-infected patients. The polyclonality was 1.5-fold lower in HIV-infected patients with CD4(+) T-cell count below 350/-microL ( P =0.007). For EBV load >1000 copies/10(6) PBMC, the levels of cell-associated EBV DNA and those of EBV-specific cellular immunity, either in intensity or in polyclonality, or both, were inversely correlated. These findings demonstrate early impairment of the EBV-specific cellular immune control with progressive increase of EBV reactivation in the course of HIV infection. These observations likely provide a basis for appreciating the risk to develop non-Hodgkin's lymphomas in HIV-infected individuals.

Background: The prevalence of antibodies to cardiolipin (a-CL) in patients with Raynaud's phenomenon (RP) and/or related disorders (rD) is not known. Objective: The purpose of this study was to determine the prevalence of these antibodies. Methods: We assayed IgG a-CL in 230 consecutive patients with RP/rD and compared the results with those in a series of 230 control blood donors. Results: Sixteen percent of patients were a-CL positive versus 7.8% of the control donors ( p = 0.014). The prevalence of a-CL positivity was 8.7% for primary RP, 10.5% for secondary RP, 8% for chilblains, 25% for essential acrocyanosis, 20% for connective tissue diseases, and 17% for undifferentiated connective tissue diseases. Among patients with digital necrosis, 24% were a-CL positive. Conclusion: The prevalence of abnormal a-CL titers is higher in patients with RP/rD than in control donors, especially in patients with a connective tissue disease. (J Am Acad Dermatol 1998;38:555-8.)

Publisher Summary This chapter discusses the methods of detection and clinical use of liver cytosol antigen type 1 autoantibodies (anti-LC-1 antibodies). Anti-LC-1 antibodies are named according to the terminology of different autoantibodies such as antimitochondrial antibodies and antimicrosome antibodies. The chapter includes three detection methods––including indirect immunofluorescence, ouchterlony double immunodiffusion, and immunoblot using rat or human liver. One of the two essential markers for autoimmune hepatitis type 2, anti-LC 1 antibodies are found in hepatitis C virus (HCV) infection; such false-positive results are eliminated by testing for HCV antibodies, HCV controlled by HCV RNA assay. A careful reading of the hepatocyte cytoplasmic fluorescence is essential for the classical detection of autoantibodies for liver diseases by indirect immunofluorescence on rat liver.

Les tests cellulaires pour l’explorationin vitro de l’hypersensibilite immediate, utilisent le polynucleaire basophile du sang peripherique. Ce leucocyte qui possede la plupart des proprietes du mastocyte, un recepteur membranaire de forte affinite pour les IgE (FceR1) et des granulations contenant des mediateurs preformes de l’anaphylaxie, est un bon temoin de la sensibilisation mastocytaire contre un allergene. Mais comme ce dernier est d’un acces difficile, pour les etudesin vitro chez l’homme on prefere travailler sur le basophile.

An autoantibody to liver cytosol was previously described in childhood autoimmune chronic active hepatitis type 2. The antigen, liver cytosol antigen type 1, was for the first time partially purified using gel filtration and ion exchange chromatography, and it was characterized using immunodiffusion, immunoblot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitate. Immunoblot detected a unique antigenic peptide at 62 kD from human cytosol and at 58 kD from rat cytosol. The same peptides were also detected when immunoprecipitates of liver cytosol antigen type 1 and autoantibodies to liver cytosol antigen were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polymeric structure, probably a tetramer, is suggested for native liver cytosol antigen type 1 because in gel filtration chromatography liver cytosol antigen type 1 was eluted as a protein of a molecular weight between 240 and 290 kD when human liver cytosol was fractionated and between 220 and 270 kD from rat liver cytosol. Liver cytosol antigen type 1 is probably poor in carbohydrates because it was not stained by periodic acid-Schiff stain. The autoantibodies to liver cytosol were frequently found in association with antiliver kidney microsomal autoantibodies type 1, which are directed against the cytochrome P-450 of the IID6 subfamily. Antiliver kidney microsomal autoantibodies type 1 but not antiliver cytosol autoantibodies were found in association with antibodies to hepatitis C virus. Autoantibodies to liver cytosol antigen type 1 seem to be a more specific marker for autoimmune hepatitis type 2 than antiliver kidney microsomal antibodies type 1 autoantibodies.

Recent studies have shown that hepatitis C virus antibodies are present in a large proportion of patients with autoimmune hepatitis type 2. We have studied 83 patients with liver/kidney microsome antibody-positive type 1 hepatitis. Hepatitis C virus antibodies were sought in every case by second-generation tests (hepatitis C virus enzyme-linked immunosorbent assay and recombinant immunoblot assay). Hepatitis C virus RNA sequences were sought in 22 patients (12 with recombinant immunoblot assay-positive results and 10 with recombinant immunoblot assay-negative results) by means of polymerase chain reaction and by use of primers located in the 5′ noncoding region. Sixty-four patients (77%) had positive results for hepatitis C virus antibodies in the enzyme-linked immunosorbent assay test, and 41 (49.3%) were confirmed by recombinant immunoblot assay. Hepatitis C virus RNA sequences were found in all the recombinant immunoblot assaypositive patients but in none of the 10 who were recombinant immunoblot assay-negative. The recombinant immunoblot assay-negative patients were younger than those who were positive (13 ± 11 vs. 50 ± 11 years) and had higher γ-globulin levels and liver/kidney microsome antibody-positive type 1 titers (61% had a titer of 1:1,000 or more, vs. only 17% of the recombinant immunoblot assay-positive patients). On the basis of these results, chronic hepatitis with liver/kidney microsome antibody-positive type 1 can be divided into two subgroups: (a) true autoimmune hepatitis type 2 (mainly observed in young women), with high titers of liver/kidney microsome antibody-positive type 1, and in which direct autoimmune mechanisms appear to be prominent; and (b) hepatitis C virus-associated hepatitis with liver/kidney microsome antibody-positive type 1 (generally affecting older patients, especially men), with low titers of liver/kidney microsome antibody-positive type 1, and in which the autoimmune process could be a consequence of hepatitis C virus infection. (HEPATOLOGY 1992;16:630–636.)

A 14-year-old girl presented with anti-liver-kidney microsome autoimmune hepatitis preceded by alopecia 3 years earlier. Both pathologies were greatly improved by immunosuppressive therapy. Alopecia is a newly reported extrahepatic manifestation of type 2 autoimmune hepatitis. Its appearance could alert the clinician to an increased risk of autoimmune hepatitis in children.

Sixty-five patients with histologically proven chronic active hepatitis of unknown cause but associated with the antiliver/kidney microsome antibody type 1, confirmed by immunofluorescence and immunoprecipitation, were selected as forming a special entity. This disease was found to be rare with a prevalence of 5/1,000,000. The female to male ratio was 8:1. The condition occurred at all ages but was most common between the ages of 2 and 14 years. In 22 of the 65 cases, the hepatitis was associated with an autoimmune disease, most commonly insulin-dependent diabetes, autoimmune thyroid disease and vitiligo. The same autoimmune diseases were present in first-degree relatives from seven families. In 36 cases, the onset of disease resembled acute viral hepatitis. Serum biochemical tests showed marked elevation in aminotransaminases and hypergammaglobulinemia. Paradoxically, serum and salivary IgA levels were often normal or low. Histologic findings were multifocal hepatic necrosis with bridging in the acute stage, and aggressive hepatitis with mononuclear cell infiltration or macronodular cirrhosis in the late stages. Serologically, apart from the presence of antiliver/kidney microsome antibody type 1, the disease was characterized by the absence of antiactin, antimitochondria and antinucleus antibodies; however, organ-specific autoantibodies were often present. The clinical course was usually severe: six patients in the acute stage presented with fulminant hepatitis, and all, except two, other patients progressed to cirrhosis. Prolonged treatment with corticosteroids and immunosuppressants was usually effective in rendering the cirrhosis inactive. The cumulative survival rate was 51% at 14 years. We propose to call this entity "anti-LKM1 chronic active hepatitis" or "autoimmune hepatitis type II" to differentiate it from classical "lupoid hepatitis" or autoimmune hepatitis type I.

A new autoantibody was detected by immunoprecipitation in the serum of 21 patients with chronic active hepatitis. The antibody reacted against a soluble cytosolic antigen in liver. The antibody was organ specific but not species specific and was therefore called anti-liver cytosol antibody Type 1 (anti-LC1). In seven of 21 cases, no other autoantibody was found; the remaining 14 cases had anti-liver/kidney microsome antibody Type 1 (anti-LKM1). With indirect immunofluorescence, a distinctive staining pattern was observed with the seven sera with anti-LC1 and without anti-LKM1. The antibody stained the cytoplasm of hepatocytes from four different animal species and spared the cellular layer around the central veins of mouse and rat liver that we have called juxtavenous hepatocytes. The immunofluorescence pattern disappeared after absorption of sera by a liver cytosol fraction. The 14 sera with both antibodies displayed anti-LC1 immunofluorescent pattern after absorption of anti-LKM1 by the liver microsomal fraction. The anti-LC1 was found in the serum only in patients with chronic active hepatitis of unknown cause. Anti-LC1 antibody was not found in sera from 100 patients with chronic active hepatitis associated with anti-actin antibody classic chronic active hepatitis Type 1, 100 patients with primary biliary cirrhosis, 157 patients with drug-induced hepatitis and a large number of patients with liver and nonliver diseases. This new antibody was considered a second marker of chronic active hepatitis associated with anti-LKM1 (anti-LKM1 chronic active hepatitis) or autoimmune chronic active hepatitis Type 2.