Kappa free light chains is a valid tool in the diagnostics of MS: A large multicenter study.

OBJECTIVE: To validate kappa free light chain (KFLC) and lambda free light chain (LFLC) indices as a diagnostic biomarker in multiple sclerosis (MS). METHODS: We performed a multicenter study including 745 patients from 18 centers (219 controls and 526 clinically isolated syndrome (CIS)/MS patients) with a known oligoclonal IgG band (OCB) status. KFLC and LFLC were measured in paired cerebrospinal fluid (CSF) and serum samples. Gaussian mixture modeling was used to define a cut-off for KFLC and LFLC indexes. RESULTS: The cut-off for the KFLC index was 6.6 (95% confidence interval (CI) = 5.2-138.1). The cut-off for the LFLC index was 6.9 (95% CI = 4.5-22.2). For CIS/MS patients, sensitivity of the KFLC index (0.88; 95% CI = 0.85-0.90) was higher than OCB (0.82; 95%CI = 0.79-0.85; p < 0.001), but specificity (0.83; 95% CI = 0.78-0.88) was lower (OCB = 0.92; 95% CI = 0.89-0.96; p < 0.001). Both sensitivity and specificity for the LFLC index were lower than OCB. CONCLUSION: Compared with OCB, the KFLC index is more sensitive but less specific for diagnosing CIS/MS. Lacking an elevated KFLC index is more powerful for excluding MS compared with OCB but the latter is more important for ruling in a diagnosis of CIS/MS.

Diagnostic thresholds for free light chains in multiple myeloma depend on the assay used.

Leukemia accepted article preview online, 20 November 2017. doi:10.1038/leu.2017.335.

The role of methylation, DNA polymorphisms and microRNAs on HLA-G expression in human embryonic stem cells.

The human leukocyte antigen (HLA)-G gene seems to play a pivotal role in maternal tolerance to the fetus. Little is known about HLA-G expression and its molecular control during in vivo human embryogenesis. Human embryonic stem cells (hESC) provide an interesting in vitro model to study early human development. Different studies reported discrepant findings on whether HLA-G mRNA and protein are present or absent in hESC. Several lines of evidence indicate that promoter CpG methylation and 3' untranslated region (3'UTR) polymorphisms may influence HLA-G expression. We investigated how HLA-G expression is linked to the patterns of promoter methylation and explored the role of the 3'UTR polymorphic sites and their binding microRNAs on the post-transcriptional regulation of HLA-G in eight hESC lines. We showed that, while the gross expression levels of HLA-G are controlled by promoter methylation, the genetic constitution of the HLA-G 3'UTR, more specifically the 14bp insertion in combination with the +3187A/A and +3142G/G SNP, plays a major role in HLA-G mRNA regulation in hESC. Our findings provide a solid first step towards future work using hESC as tools for the study of early human developmental processes in normal and pregnancy-related disorders such as preeclampsia.

Belgian recommendations on ANA, anti-dsDNA and anti-ENA antibody testing.

Autoantibodies to nuclear antigens, i.e. antinuclear antibodies (ANA), antibodies to double-stranded DNA (dsDNA) and extractable nuclear antigens (ENA), are useful as diagnostic markers for a variety of autoimmune diseases. In March 2010, the Belgian national External Quality Assessment Scheme sent a questionnaire on ANA, anti-dsDNA and anti-ENA antibody testing designed by the Dutch EASI (European Autoimmunity Standardization Initiative) team, to all clinical laboratories performing ANA testing. Virtually all laboratories completed the questionnaire (97·7%, 127/130). This paper discusses the results of this questionnaire and provides valuable information on the state-of-the-art of ANA, anti-dsDNA and anti-ENA antibody testing as practiced in the Belgian laboratories. In addition, this work presents practical recommendations developed by the members of the advisory board of the scheme as a result of the outcome of this study.

Accuracy of soluble human leukocyte antigen-G for predicting pregnancy among women undergoing infertility treatment: meta-analysis.

BACKGROUND: There have been concerns about validity and accuracy of the measurement of sHLA-G in embryo culture supernatants. In this systematic review, we quantified the diagnostic accuracy of sHLA-G for predicting the ability to achieve clinical pregnancy in women who are undergoing infertility treatment. METHODS: Medline and Embase were searched up to 7 September 2007, for full English and non-English articles concerning cohort studies evaluating sHLA-G in embryo culture for predicting clinical pregnancy in women undergoing IVF and ICSI. RESULTS: Eleven studies including 1813 patients met our inclusion criteria. In the individual studies, sensitivity ranged from 0.01 to 0.97, specificity from 0.18 to 0.98, the positive likelihood ratio from 0.34 to 3.21 and the negative likelihood ratio from 0.08 to 1.01. These values were highly heterogeneous with, in each case, I(2) values of >75%, and P-values for the Q statistic of <0.001, arguing against generating a pooled estimate for these diagnostic test properties. The diagnostic odds ratios (DORs) ranged from 0.92 to 24.82 in the individual studies with an I(2) value of 49% indicating moderate heterogeneity. Therefore, the meta-analysis combined the logs of the DORs, which are derived from sensitivity and specificity. A random-effects model yielded a summary DOR of 4.38 (95% CI, 2.93-6.55), consistent with modest diagnostic accuracy. Interestingly, an a priori defined subgroup analysis restricted to six studies with good quality embryos showed a better diagnostic performance with a DOR of 12.67 (95% CI, 3.66-43.80) to predict the ability to achieve clinical pregnancy in women undergoing infertility treatment. CONCLUSIONS: Further research is needed with single-embryo culture, single-embryo transfer and highly sensitive detection techniques to determine the potential application of measuring sHLA-G in culture supernatant.

A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection.

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.

Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR.

We have identified a novel 529bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10(2)-10(4) tachyzoites and 100-3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.

DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice.

C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-gamma) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-gamma. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.

Opsonization of Toxoplasma gondii tachyzoites with nonspecific immunoglobulins promotes their phagocytosis by macrophages and inhibits their proliferation in nonphagocytic cells in tissue culture.

We have recently shown that Toxoplasma gondii tachyzoites grown in in vitro culture can bind unspecific immunoglobulin (Ig) through their Fc moiety. We show now that Fc receptors are also present on T. gondii within the host animal, and that intraperitoneal parasites in immunocompetent mice are saturated with unspecific Ig. We have also investigated the effect of the parasite's Fc receptor on the interaction of tachyzoites with mammalian cells, using the Vero cell line as a model for nonphagocytic host cells and murine peritoneal macrophages in primary culture as a model for phagocytic cells. Coating of tachyzoites with parasite-unrelated Ig did not enhance their invasive capacity in either target cell type, but slightly decreased the parasite proliferation. Moreover, phagocytosis by macrophages was increased by approximately 50% when parasites were coated with unspecific Ig. These results indicate that the Fc receptor on T. gondii affects the balance between invasion and phagocytosis in a way that is detrimental to the parasites.

Evaluation of recombinant dense granule antigen 7 (GRA7) of Toxoplasma gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain.

Dense granule protein 7 (GRA7) of Toxoplasma gondii was expressed in Escherichia coli as a fusion protein. The leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues. After purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG). For two sets of IgG-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was obtained. For chronic-phase sera, the sensitivity of detection was 79%, but chronic-phase sera with low titers were more difficult to detect (65% sensitivity for sera with immunofluorescence titer of 1/64). When GRA7 was combined with Tg34AR (rhoptry protein 2 C-terminal fragment), the sensitivity rose to 96%. For a set of acute-phase serum samples tested on GRA7, the sensitivity of detection was 94%, and high-titer IgM-positive sera were detected at an especially high rate. In contrast, when Tg34AR was used, the sensitivity was only 85% for this latter set of serum samples. Three truncated GRA7 fragments containing the same leader peptide as that of recombinant GRA7 were produced. The shortest fragment (97 N-terminal amino acids) was not reactive with human sera or with a specific anti-GRA7 monoclonal antibody, while the two larger fragments were reactive. The most important antigenic domain of GRA7 for human sera was localized between residues 97 and 146. The epitope for the specific monoclonal antibody could be further narrowed down by the use of synthetic peptides, but this epitope is not recognized by sera from T. gondii-infected humans. These results indicate that GRA7 may be considered as an additional tool for studying the immune response to T. gondii.

Acute and chronic phases of Toxoplasma gondii infection in mice modulate the host immune responses.

Murine antibody responses to soluble proteins are generally restricted to the immunoglobulin G1 (IgG1) isotype. When mice were infected with Toxoplasma gondii Beverley and concomitantly immunized with a soluble unrelated protein antigen, a modification in the isotypic distribution of antibodies directed against this nonparasite antigen was observed, with a preferential production of IgG2a. Interestingly, when mice were immunized with a soluble protein antigen during the chronic phase (day 40) of infection with T. gondii Beverley, a similar modification in the isotypic distribution of antiprotein antibodies was observed.

Identification and characterization of a Fc receptor activity on the Toxoplasma gondii tachyzoite.

The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite.

Comparison of the internal transcribed spacer, ITS 1, from Toxoplasma gondii isolates and Neospora caninum.

The internal transcribed spacer (ITS1) region and the 5' part of the 5.8S ribosomal RNA gene of the ribosomal DNA repeat from 20 Toxoplasma gondil isolates was sequenced and found to be identical in all isolates, independent of host origin or virulence to mice. The ITS1 region from the closely related coccidian parasite Neospora caninum differed in 22% of its nucleotides. Hence, the ITS1 region provides a good marker for the distinction of T. gondii and N. caninum but is not useful for epidemiology studies of T. gondii.

Iron and total iron-binding capacity in serum of patients receiving iron-dextran: Kodak Ektachem methodologies, spectrophotometry, and atomic-absorption spectrometry compared.

We compared the analytical performance of the Kodak Ektachem XR700 assays of iron (Fe) and total iron-binding capacity (TIBC) with that of a conventional ferrozine assay (performed with a Cobas-Bio) and occasionally with that of atomic-absorption spectrometry (AAS). The correlation was modest between Kodak and Cobas-Bio concentrations of Fe in serum (r = 0.79). Multiple outliers were noticed in samples from hemodialysis patients, with Cobas values exceeding those of Kodak by as much as 26 mumol/L. These intermethod differences were not dissipated by dialysis, but were invariably accompanied by an even higher total Fe content of the serum as judged by AAS (20 to 80 mumol/L higher). TIBC values by Kodak and Cobas-Bio were highly correlated (r = 0.99); again, the Cobas-Bio results exceeded those by Kodak in some hemodialysis patients, but always for samples with higher Fe concentrations by the Cobas-Bio. By AAS, the TIBC values of these patients also exceeded those by Kodak, to about the same extent as observed for serum Fe. These intermethod differences in Fe and TIBC were seen only in patients who had received an intravenous Fe-dextran (Imferon) injection two to three days before blood sampling but could be generated in vitro by adding Imferon to serum from normal controls. Less than 6% of dextran-bound Fe is measured as Fe by Kodak, as opposed to 20-30% by Cobas-Bio and 89-120% by AAS. We conclude that the Kodak Fe slides are superior to liquid reagents, by exclusively measuring protein-bound circulating Fe pools.

Cell surface antibodies in type 1 (insulin-dependent) diabetic patients. I. Presence of immunoglobulins M which bind to rat pituitary cells.

A standardized method has been developed for the assay of cell surface antibodies in IgM- and IgG-fractions from human serum. Suspensions of adult rat islet B cells, islet non-B cells, and anterior pituitary cells were used as antigen source and a cell sorter as analyser of the immunoglobulin binding to individual cells. Assay conditions were selected wherein no surface antibodies were detected in 33 control subjects younger than 20 years. In 30% of Type 1 (insulin-dependent) diabetic patients, surface antibodies were measured with rat anterior pituitary cells as well as with rat islet B cells. Binding to pituitary cells occurred with IgM- and IgG-fractions and correlated positively with IgG binding to islet B cells. At onset of the disease, the prevalence of IgM-rat anterior pituitary cell surface antibodies was higher than that of IgG-rat anterior pituitary cell surface antibodies. Cell surface antibodies were also detected in first-degree relatives of Type 1 diabetic patients, but corresponded primarily to IgM-rat anterior pituitary cell surface antibodies. It is concluded that the development of Type 1 diabetes in subjects younger than 20 years is associated with the generation of both IgM and IgG cell surface antibodies. The IgM surface antibodies may result from stimulated production of polyreactive natural autoantibodies and could precede the switch to the formation of monoreactive IgG autoantibodies. The assay of IgM cell surface antibodies can be useful in studies on the sequence of immune events in diabetes and other autoimmune disease.

Cell surface antibodies in type 1 (insulin-dependent) diabetic patients. II. Presence of immunoglobulins M which bind to lymphocytes.

A standardized cell surface antibody assay was used to measure binding of circulating human immunoglobulins to rat or piglet splenocytes. In 100-fold diluted serum fractions, lymphocyte surface antibodies were detected in 30% of Type 1 (insulin-dependent) diabetic patients under 20 years of age but in none of 33 control subjects. Binding occurred with T and B lymphocytes, appeared unrelated to Fc receptors or protein glycosylation and was not attributable to insulin or albumin antibodies. At clinical onset of the disease, the lymphocyte surface antibodies belonged primarily to the IgM-class. Their presence was positively correlated to that of IgM-pituitary cell surface antibodies and their absorption by anterior pituitary cells occurred as well as by splenocytes. Lymphocyte surface antibodies remained present during the first years of insulin treatment. They were also detected in first degree relatives of lymphocyte surface antibody-positive patients. It is unlikely that IgM-lymphocyte surface antibodies mark the destructive process in the pancreatic B cell population. They may, instead, express a state of immune reactivity which precedes the formation of IgG-autoantibodies and therefore be associated with an event in the development of diseases such as Type 1 (insulin-dependent) diabetes.

First trimester prenatal diagnosis of lysosomal storage disease. Study of alpha-L-fucosidase isoenzyme patterns in fetal and maternal tissue.

The normal range of activities of 6 lysosomal enzymes was determined in extracts of chorionic villi samples obtained by a rigid forceps in the first trimester of pregnancy. These activities were compared to those in villi obtained after abortion and in cultured amniotic fluid cells and fibroblasts. For five of the six enzymes tested, the data suggest that first trimester prenatal diagnosis should be possible and reliable. For the sixth enzyme, alpha-L-fucosidase, where on occasion very low activities were found, the results obtained on fresh chorionic villi have to be interpreted with extreme caution. Considerable lysosomal enzyme activities were also found in maternal decidua. Therefore, extreme care must be taken in the preparation of chorionic villi for prenatal diagnosis of lysosomal disorders since even small amounts of maternal tissue could lead to misdiagnosis. This study has allowed us to monitor 2 pregnancies at risk for lysosomal storage disease. Differences in the isoenzyme pattern of alpha-L-fucosidase were found in chorionic villi and maternal decidua. Although further studies are required, this observation could lead to the development of immuno-biochemical methods to evaluate the purity of chorionic villi used for prenatal diagnosis.