J. Haas

L-Selectin is a possible biomarker for individual PML risk in natalizumab-treated MS patients

Objective: To find biomarkers identifying patients at risk for the development of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. Methods: Patients were recruited from 10 European and US cohorts. Of 289 patients with multiple sclerosis (MS), 224 had been treated with natalizumab (18–80 months), 21 received other immune-modulatory treatments, and 28 were untreated. We had access to samples from 16 natalizumab PML patients. Eight of these patients had given blood before the diagnosis of PML. We also analyzed non-natalizumab-treated patients who developed PML (n = 10) and age- and sex-matched healthy donors (n = 31). All flow cytometric assessments were done on previously cryopreserved, viable peripheral blood mononuclear cells. Results: The percentage of l-selectin-expressing CD4+ T cells was significantly lower in patients treated long-term with natalizumab (40.2%) when compared with patients not receiving natalizumab treatment (47.2%; p = 0.016) or healthy controls (61.0%; p < 0.0001). An unusually low percentage (9-fold lower; 4.6%) was highly correlated with the risk of developing PML in the patient group with available pre-PML samples when compared with non-PML natalizumab-treated patients (p ≤ 0.0001). Samples were gathered between 4 and 26 months before PML diagnosis. Conclusions: The cell-based assessment of the percentage of l-selectin-expressing CD4 T cells could provide an urgently needed biomarker for individual PML risk assessment.

Glatiramer acetate improves regulatory T-cell function by expansion of naive CD4(+)CD25(+)FOXP3(+)CD31(+) T-cells in patients with multiple sclerosis.

Naturally occurring regulatory T-cells (Treg) exhibit impaired function in patients with relapsing-remitting multiple sclerosis (RRMS) resulting from an age-inappropriate disproportion between prevalences of newly generated CD31-coexpressing naive Treg and long-lived memory Treg in the periphery. Recent evidence suggests that the immunomodulatory action of glatiramer acetate (GA) includes effects on Treg function and frequencies. We prospectively assessed suppressive activities and frequencies of Treg and Treg subsets in 15 patients with RRMS undergoing long-term therapy with GA. Treatment for up to six months reconstituted naive Treg and increased total Treg numbers with concomitant reversion of the Treg defect.

Interferon Beta–Induced Restoration of Regulatory T-Cell Function in Multiple Sclerosis Is Prompted by an Increase in Newly Generated Naive Regulatory T Cells

BACKGROUND Naturally occurring regulatory T (T(reg)) cells are functionally impaired in patients with relapsing-remitting multiple sclerosis. We recently showed that prevalences of newly generated CD31-coexpressing naive T(reg) cells (recent thymic emigrant-T(reg) cells) are critical for suppressive function of circulating T(reg) cells, and a shift in the homeostatic composition of T(reg)-cell subsets related to a reduced de novo generation of recent thymic emigrant-T(reg) cells may contribute to the multiple sclerosis (MS)-related T(reg)-cell dysfunction. Interferon beta, an immunomodulatory agent with established efficacy in MS, lowers relapse rates and slows disease progression. Emerging evidence suggests that T(reg)-cell suppressive capacity may increase in patients with MS undergoing treatment with interferon beta, although the mechanisms mediating this effect are uncertain. OBJECTIVE To evaluate the effect of interferon beta treatment on the suppressive activity and the homeostasis of circulating T(reg) cells in patients with MS. PARTICIPANTS Twenty patients with relapsing-remitting MS and 18 healthy control subjects. INTERVENTIONS Administration of interferon beta. MAIN OUTCOME MEASURES Effect of interferon beta on T(reg)-cell homeostasis and suppressive capacity. RESULTS Suppressive capacities of T(reg) cells were consistently upregulated at 3 and 6 months after treatment with interferon beta. The restoration of T(reg)-cell function was paralleled by increased naive recent thymic emigrant-T(reg) cells and a coincidental reduction in memory T(reg) cells. CONCLUSION The increase in T(reg)-cell inhibitory capacity mediated by interferon beta treatment can be explained by its effect on the homeostatic balance within the T(reg) cell compartment.

Mutations in the COL5A1 Coding Sequence Are Not Common in Patients With Spontaneous Cervical Artery Dissections

BACKGROUND AND PURPOSE The dermal connective tissue of most patients with spontaneous cervical artery dissections (sCAD) contains abnormal collagen fibers. This suggests a predisposing connective tissue defect. The ultrastructural abnormalities in the skin of patients with sCAD have similarity with the morphological alterations in patients with Ehlers-Danlos syndrome type II, a dominant hereditary disorder that has been correlated in some patients to mutations within the genes encoding type V collagen. The aim of this study was to assess the alpha 1 chain of type V collagen (COL5A1) as a candidate gene for sCAD. METHODS We searched for mutations in the COL5A1 gene in cDNA from cultured fibroblasts of 19 patients with sCAD using single-strand conformational polymorphism analysis and nucleotide sequence analysis of polymerase chain reaction-amplified fragments of the whole COL5A1 coding sequence. RESULTS We detected 1 missense mutation leading to a predicted amino acid (192D/N) substitution within the N-terminal propeptide in 2 siblings. All other patients showed regular COL5A1 sequences with some silent polymorphisms. CONCLUSIONS Mutations in the COL5A1 gene do not appear to be a major factor in the etiology of sCAD.

Diagnosis of cytomegalovirus encephalitis in patients with AIDS by quantitation of cytomegalovirus genomes in cells of cerebrospinal fluid

A nested polymerase chain reaction (PCR) assay was used to determine the levels of cytomegalovirus (CMV) genomes in cells of CSF from 19 patients with AIDS and 12 human immunodeficiency virus type I (HIV-1) seronegative individuals with various neurologic disorders. Five AIDS patients had autopsy-proven CMV encephalitis (CMVE) and 14 patients had no evidence of CMV-related CNS manifestations. CSF cells from AIDS patients with confirmed CMVE harbored viral genomes at a median value of 3,333/105 cells(range, 1,667 to 5,333/105 cells; mean, 3,558/105 cells) compared with a median value of 125/105 cells (range, 9 to 1,000/105 cells; mean, 281/105 cells) for AIDS patients with CMV-unrelated symptoms and a median value of 19/105 cells (range, 0 to 562/105 cells; mean, 52/105 cells) for HIV-1 seronegative control subjects. A subset of CSF samples was assessed using a modified single round amplification PCR with a detection limit of 500 viral copies. CMV DNA was detected in all four specimens from AIDS patients with proven CMVE, in two of five AIDS patients without CMVE, and in none of five seronegative control subjects. Quantitation of CMV genomes in CSF cells is indicative of latent or productive CMV infection and is a reliable means for diagnosis of CMVE in patients with AIDS. Detection of a cutoff value of cellular CMV genomes by means of nonquantitative PCR may identify patients at risk for CMV infection of the CNS.

Single-cell PCR analysis of the immunoglobulin heavy-chain CDR3 region for the diagnosis of leptomeningeal involvement of B-cell malignancies using standard cerebrospinal fluid cytospins

The diagnosis of leptomeningeal B-cell malignancies is based on the identification of malignant B cells in the cerebrospinal fluid (CSF). We have established a polymerase chain reaction (PCR) approach to characterize the clonally diverse gene encoding the immunoglobulin heavy-chain (IgH) third complementarity determining region (CDR3) of single B cells. We demonstrate that single-cell PCR is readily applicable to individual cells derived from routine CSF cytospins and is a powerful method to discriminate monoclonal neoplastic from polyclonal reactive B-cell responses. Single-cell PCR analysis, as a new tool for the diagnosis and monitoring of neoplastic meningitis associated with B-cell malignancies, is particularly important if cytology, immunocytochemistry, flow cytometry and automated gene scanning of CSF samples are unable to detect malignant monoclonal proliferation.

Rapid distinction of acute demyelinating disorders and central nervous system lymphoma by molecular analysis of cerebrospinal fluid cells.

Abstract Polymerase chain reaction (PCR) based automated high-resolution fragment analysis of rearranged immunoglobulin heavy-chain genes is a highly sensitive means for identifying clonal B-cell responses. We used this technique to distinguish polyclonal inflammatory from monoclonal neoplastic B-cell populations in the cerebrospinal fluid (CSF) of three patients with acute demyelinating disorders of the central nervous system whose clinical, magnetic resonance imaging (MRI) and CSF features did not permit unequivocal exclusion of primary central nervous system lymphoma (pC-NSL). This approach is highly suitable for detecting CNS inflammation particularly when lymphomatous involvement cannot be ruled out by noninvasive diagnostic procedures alone.

Leptomeningeale Aussaat einer chronisch lymphatischen Leukämie Molekulargenetischer Nachweis im Liquor

ZusammenfassungDie Diagnose einer leptomeningealen Infiltration ist bei der chronisch lymphatischen Leukämie (CLL) mit konventionellen zytomorphologischen Methoden nicht hinreichend möglich. Das Zellbild ist meist monomorph, und eindeutige Malignitätskriterien fehlen. Eine Immunphänotypisierung mit Bestimmung von Leukozytendifferenzierungsantigenen erlaubt eine weitere Eingrenzung, ist jedoch häufig wegen geringen Zellmaterials nur eingeschränkt möglich. Molekulargenetische Methoden können zur weiteren Diagnosesicherung eingesetzt werden. Bei einem 54jährigen Patienten mit einer seit 5 Jahren bestehenden Diagnose einer CLL konnte trotz klinischen Verdachts weder kernspintomographisch noch in der konventionellen Liquordiagnostik ein Anhalt für eine leptomeningeale Infiltration der CLL gefunden werden. Mit der Polymerase-Kettenreaktion (PCR) wurde die hochvariable, B-Zell-Klon-spezifische CDR3-Region des für die Immunglobulinkette-Schwerkette (IgH) kodierenden Locus selektiv amplifiziert. Als Ausgangsmaterial wurde zelluläre DNA aus Liquor und Blut des Patienten verwendet. Die Analyse der PCR-Produkte mit hochauflösender Gelelektrophorese ergab sowohl für B-Zellen aus dem Liquor als auch für B-Zellen aus dem Blut ein einzelnes DNA-Fragment. Hierdurch wurde der Nachweis erbracht, daß die Zellpopulationen in beiden Kompartimenten monoklonal sind. Die DNA-Sequenz-Analyse der amplifizierten CDR3-Segmente bestätigte die klonale Identität der Zellen und damit eindeutig die leptomeningeale Infiltration der CLL.SummaryThe diagnosis of leptomeningeal dissemination of chronic lymphatic leukemia (CLL) by conventional cytology is unreliable because cytomorphologic criteria of malignancy are often lacking. Immunophenotyping of leukocyte differentiation antigens may also be of limited diagnostic value due to the small number of cells in cerebrospinal fluid (CSF) samples. Molecular methods may support the specific diagnosis of leptomeningeal infiltration of CLL. We present an 54 old patient who was diagnosed with CLL five years ago. Despite clinical signs of leptomeningeal involvement neither magnetic resonance imaging (MRI) nor conventional CSF analysis were suggestive of lymphomatous meningitis. Using PCR we selectively amplified the highly variable and clone-specific CDR3 region of the locus encoding the immunoglobulin heavy chain (IgH) in DNA obtained from both CSF and peripheral blood cells. Analysis of PCR products by high resolution gel electrophoresis revealed a single DNA fragment respectively indicating the presence of a monoclonal cell population in both compartments. DNA sequence analysis of the amplified CDR3 segments confirmed the clonal identity of cells and the leptomeningeal dissemination of CLL.

Leptomeningeale Tumorzellinfiltration als Erstmanifestation eines Immunozytoms (M. Waldenström)

ZusammenfassungDas Immunozytom (M. Waldenström, Makroglobulinämie Waldenström) ist eine seltene, chronisch lymphoproliferative Erkrankung der B-Zell-Reihe. Die Erkrankung ist charakterisiert durch erhebliche Mengen zirkulierender monoklonaler Immunglobuline der Klasse IgM und eine lymphoplasmozytoide Knochenmarksinfiltration. Eine Beteiligung des peripheren Nervensystems ist häufig und äußert sich klinisch als Polyneuropathie (5–10%). Ein isolierter leptomeningealer Befall durch neoplastische Zellen ist sehr selten und wurde bislang nur in einzelnen Fällen berichtet. Die Diagnosestellung ist schwierig, insbesondere wenn die Liquorzytologie nicht eindeutig ist. Im vorliegenden Fall handelt es sich um einen Patienten mit neu aufgetretener Wesensänderung und kognitiven Defiziten. Der initiale Liquorzellbefund war kompatibel mit einer chronisch lymphozytären Meningitis. Die serologische Detektion einer IgM-Paraproteinämie sowie die Knochenmarkszytologie wiesen auf das Vorliegen eines Immunozytoms hin. Die selektive Untersuchung der B-Zell-Klonalität mittels Polymerasenkettenreaktion (PCR)-basierter Amplifikation der rekombinierten CDR3-Region des IgH-Gens sowohl im Zelllysat des Gesamtliquors als auch in individuellen Liquorzellen wies eine monoklonale B-Zell-Population und damit eine leptomeningeale Tumorzellinfiltration durch das Immunozytom nach.AbstractImmunocytoma (Waldenström’s macroglobulinemia) is a rare chronic lymphoproliferative disorder of B-cell origin. It is characterized by the presence of large amounts of circulating monoclonal immunoglobulin M (IgM) and lymphoplasmocytoid bone marrow infiltration. Affection of the peripheral nervous system is common and causes polyneuropathy (5–10%). An isolated leptomeningeal infiltration by neoplastic cells is very rare and has been reported in few cases only. The diagnosis is difficult, in particular if cerebrospinal fluid (CSF) cytology is inconclusive. We present the case of a patient who developed a personality disorder and cognitive impairment. Initial CSF findings were compatible with chronic lymphocytic (aseptic) meningitis. The serologic detection of IgM paraproteinemia and bone marrow cytology suggested immunocytoma. The selective analysis of B-cell clonality in both whole CSF cell lysates and individual CSF cells using polymerase chain reaction (PCR) based amplification of the rearranged CDR3 region of the IgH gene revealed the presence of a monoclonal B-cell population and was diagnostic for leptomeningeal tumor cell infiltration by immunocytoma.

Wertigkeit der Polymerase-Kettenreaktion (PCR) für die Diagnostik der tuberkulösen Meningitis

Zusammenfassung,alsein ubiquitärer Keim mit geringer Pathogenität, Untersuchungsproben verunreinigen kann und die Verwendung von Primerpaaren, die gruppenspezifische mykobakterielle DNA erkennen, zu falsch-positiven Resultaten führen kann. Für die Validität der PCR ist daher neben der Sensitivität der Nachweisreaktion, die Spezifität der eingesetzten PCR-Primer von großer Bedeutung, um zwischen mykobakteriellen Subtypen zu differenzieren.Value of the polymerase chain reaction (PCR) for diagnosing tuberculous meningitisSummary