High interindividual variability in the CD4/CD8 T cell ratio and natalizumab concentration levels in the cerebrospinal fluid of patients with multiple sclerosis.

Strongly decreased leucocyte counts and a reduced CD4/CD8 T cell ratio in the cerebrospinal fluid (CSF) of natalizumab (NZB)-treated multiple sclerosis (MS) patients may have implications on central nervous (CNS) immune surveillance. With regard to NZB-associated progressive multi-focal leucoencephalopathy, we aimed at delineating a relationship between free NZB, cell-bound NZB, adhesion molecule (AM) expression and the treatment-associated shift in the CSF T cell ratio. Peripheral blood (PB) and CSF T cells from 15 NZB-treated MS patients, and CSF T cells from 10 patients with non-inflammatory neurological diseases and five newly diagnosed MS patients were studied. Intercellular adhesion molecule-1 (ICAM-1), leucocyte function antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), NZB saturation levels, and T cell ratios were analysed by flow cytometry. NZB concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Lower NZB saturation levels (P<0.02) and a higher surface expression of ICAM-1 and LFA-1 (P<0.001) were observed on CSF CD8 T cells. CSF T cell ratios (0.3-2.1) and NZB concentrations (0.01-0.42 µg/ml) showed a pronounced interindividual variance. A correlation between free NZB, cell-bound NZB or AM expression levels and the CSF T cell ratio was not found. Extremely low NZB concentrations and a normalized CSF T cell ratio were observed in one case. The differential NZB saturation and AM expression of CSF CD8 T cells may contribute to their relative enrichment in the CSF. The reduced CSF T cell ratio appeared sensitive to steady-state NZB levels, as normalization occurred quickly. The latter may be important concerning a fast reconstitution of CNS immune surveillance.

Natalizumab saturation: biomarker for individual treatment holiday after natalizumab withdrawal?

BACKGROUND: More and more patients with multiple sclerosis (MS) switch from natalizumab to fingolimod because of the risk of progressive multifocal leukoencephalopathy. The duration of the treatment holiday is still under debate referring to a possible recurrence of disease activity. AIM OF THE STUDY: The aim of this study was to evaluate the prognostic value of natalizumab saturation on T cells for the recurrence of clinical and radiological disease activity. METHODS: Cell surface-bound natalizumab saturation (in%) of CD8+ and CD4+ T cells from five patients with MS was determined before initiation of fingolimod by flow cytometry and related to clinical and MRI outcome during a 6-month follow-up. RESULTS: In two patients with either clinical or radiological disease activity, the natalizumab saturation on CD8+ and CD4+ T cells was <30%. In contrast, the remaining three patients with absence of disease activity had a median natalizumab saturation of 70% (range 59-79%) on CD4+ and 66% (range 52-68%) on CD8+ T cells. CONCLUSIONS: The data of this pilot study indicate that clinical and radiological disease activity is closely linked to natalizumab saturation at the time point of switch. The determination of natalizumab saturation may be an essential tool to monitor cessation of natalizumab treatment.

Glatiramer acetate attenuates the pro-migratory profile of adhesion molecules on various immune cell subsets in multiple sclerosis.

An altered expression pattern of adhesion molecules (AM) on the surface of immune cells is a premise for their extravasation into the central nervous system (CNS) and the formation of acute brain lesions in multiple sclerosis (MS). We evaluated the impact of glatiramer acetate (GA) on cell-bound and soluble AM in the peripheral blood of patients with relapsing-remitting MS (RRMS). Fifteen patients treated de novo with GA were studied on four occasions over a period of 12 months. Surface levels of intracellular cell adhesion molecule (ICAM)-1, ICAM-3, lymphocyte function-associated antigen (LFA)-1 and very late activation antigen (VLA)-4 were assessed in T cells (CD3(+) CD8(+) , CD3(+) CD4(+) ), B cells, natural killer (NK) cells, natural killer T cells (NK T) and monocytes by five-colour flow cytometry. Soluble E-selectin, ICAM-1, ICAM-3, platelet endothelial cell adhesion molecule (PECAM)-1, P-selectin and vascular cell adhesion molecule (VCAM)-1 were determined with a fluorescent bead-based immunoassay. The pro-migratory pattern in RRMS was verified by comparison with healthy controls and was characterized by up-regulation of LFA-1 (CD3(+) CD4(+) T cells, B cells), VLA-4 (CD3(+) CD8(+) T cells, NK cells), ICAM-1 (B cells) and ICAM-3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA-1 (CD3(+) CD4(+) ) and VLA-4 (CD3(+) CD4(+) , CD3(+) CD8(+) , NK, NK T, monocytes). Further effects included lowering of ICAM-1 and ICAM-3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro-migratory expression profile of cell-bound AM is altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects.

Isolated leptomeningeal infiltration of a primary CNS B-cell lymphoma diagnosed by flow cytometry and confirmed by necropsy.

BACKGROUND: The diagnosis of the isolated leptomeningeal involvement of a primary central nervous system B-cell lymphoma without parenchyma lesions may be difficult. Patients with leptomeningeal meningeosis lymphomatosa can present with various neurologic deficits. AIMS OF THE STUDY: To demonstrate the impact of cerebrospinal fluid (CSF) flow cytometry in the diagnosis of an isolated leptomeningeal manifestation of B-cell lymphoma by presenting an interesting case report. METHODS: Flow cytometric analysis of B-cell monoclonality of the CSF was performed as complementary diagnostic procedure in addition to CSF cytology. Final diagnosis was confirmed by necropsy. RESULTS: We suspected isolated leptomeningeal manifestation of B-cell lymphoma with palsy of the VI and VII cranial nerves in a 79-year-old male, because of mononuclear pleocytosis in CSF. Interestingly, the decisive diagnostic hint was given by implementation of flow cytometry of the CSF. Diagnosis was confirmed by postmortem autopsy. CONCLUSION: Our case shows that flow cytometry of the CSF in addition to conventional CSF cytology has the potential to accelerate diagnosis of lymphomeningeal infiltration of B-cell lymphoma.

Molecular evidence of transient therapeutic effectiveness of natalizumab despite high-titre neutralizing antibodies.

Natalizumab is a humanized monoclonal antibody directed against the alpha-4 integrin subunit of very late activation antigen-4 (VLA-4). Natalizumab neutralizing antibodies (NAB) have been found to significantly reduce beneficial effects of natalizumab treatment in multiple sclerosis. We investigated interactions of NAB with natalizumab by serial measurements of alpha-4 integrin levels on peripheral blood mononuclear cells using flow cytometry. In addition, serum concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1), the endothelial ligand of VLA-4, and serum NAB were serially determined. Natalizumab infusion led to a transient reduction in alpha-4 integrin levels on immune cells and serum sVCAM-1 levels along with serum negativity of NAB lasting for a few days post-infusion. Apparently, the high-dose effect of freshly infused natalizumab resulted in a transient neutralization of NAB possibly involving a transient therapeutic effectiveness.

Adhesion molecules are promising candidates to establish surrogate markers for natalizumab treatment.

BACKGROUND: Natalizumab is the first monoclonal antibody therapy approved for multiple sclerosis (MS). Its therapeutic mechanism is the blockade of the α4-integrin subunit of the adhesion molecule (AM) very late activation antigen-4 (VLA-4), which leads to an inhibition of immune cell extravasation into the central nervous system (CNS). METHODS: We investigated changes in the expression levels of unblocked α4-integrin and further AM (intercellular adhesion molecule-1, -2, -3 (cICAM-1, -2, -3), leukocyte function associated antigen-1 (LFA-1)) on peripheral blood mononuclear cells (PBMC) determined by flow cytometry from 25 patients with MS before the first natalizumab infusion and before the fourth infusion. In 15 MS patients AM expression was evaluated every 3 months over 1 year. RESULTS: We found a significant decrease (p < 0.0001) of unblocked α4-integrin cell surface expression on all investigated PBMC subsets (T cells -61.7%, B cells -69.1%, monocytes/macrophages -46.4%) in the blood of MS patients after 3 months of natalizumab treatment. Moreover, a continuous decrease (p < 0.05) of unblocked α4-integrin expression levels was seen after 3, 6, 9, and 12 months. As a secondary effect, expression levels of the other investigated AM were differentially affected. CONCLUSIONS: Results show a sustained decrease of unblocked α4-integrin expression not only in all patients but also in all investigated PBMC subsets. This probably results in a continuously decreasing transmigration of PBMC into the CNS and may explain the improved clinical efficacy in the second treatment year and also the increasing risk of progressive multifocal leukoencephalopathy during long-term natalizumab therapy. We conclude that AM expression profiles are promising candidates for the development of a biomarker system to determine both natalizumab treatment response and patients at risk for opportunistic CNS infections.

[Expert assessment of initial surgical management after experimental chemical eye burn]. / Begutachtung der chirurgischen Erstversorgung nach experimenteller Verätzung.

BACKGROUND: Initial surgical treatment of severe eye burns is important for the long lasting healing period. To look closer at the efficacy of the early surgical treatment and the possible consequences for woundhealing, the surgical interventions were examined in an animal experiment. MATERIALS AND METHODS: The right eye of 20 rabbits each was burned with 1 n NaOH for 30 seconds followed by a surgical treatment 48 hours later. In the first study necrosectomy, tenonplasty, application of an artificial epithelium and blepharoplasty was withdrawn and the fornices were prevented from cicatrization by mechanical treatment treatment with blunt spatula. Antiinflammatory depotinjection of Volon A 40 was applicated additionally. After an interval of 4-18 weeks the eyes were examined histologically. RESULTS: A higher risk for infections could be observed in eyes which underwent blepharorhaphy. Furthermore proliferative processes and corneal ulcerations were predominant. In contrast to these findings the latter complications could be prevented due to the clinical inspection of the artificial epithelium and described mechanical irritation of the fornices in the second study. DISCUSSION: It could be shown that proliferative processes are major complications after severe burns of the eye. Nevertheless the intensity could be minimized by the consequent therapy. Furthermore it could be demonstrated that the artificial epithelium is an effective tool providing good prognosis for subsequently planned keratoplasty. To enable necessary postoperative inspection blepharoplasty should be avoided.

Serum LH levels in the differential diagnosis of hirsute anovulatory women.

Polycystic ovarian syndrome (PCO) occurs frequently in hirsute patients. A dissociated explosive response of LH to LHRH administration has been associated with the diagnosis of PCO. Twenty-four of 58 women seen because of hirsutism were found to have ovarian dysfunction based on clinical signs such as anovulation and irregular menstrual cycles. Plasma androgen levels were elevated in the patient group. The LHRH test (200 micrograms, iv) was applied to the 24 patients and compared with 13 normal ovulatory controls. Serum FSH levels before and after the LHRH test were normal in all patients. Two patterns of LH response to LHRH stimulation were observed: an explosive response in 17 patients (delta LH: 39.4 +/- 21.8 IU/l, control group: 7.35 +/- 4.4 IU/l, P < 0.01) and a normal response in 7 patients (delta LH: 7.53 +/- 2.41 IU/l). There was a significant correlation (r = 0.63, P < 0.05) between basal LH levels and LH response to LHRH. Sensitivity and specificity calculated for basal LH levels higher than 6.0 IU/l, considering the LHRH test as reference, were 58% and 85%, respectively. The positive predictive value measuring the possibility of LH higher than 6.0 IU/l to be from a patient with PCO (explosive response to LHRH) was 92%. These data suggest that, in hirsute anovulatory patients, basal LH levels may be a good predictor in the diagnosis of polycystic ovarian syndrome.

Serum LH levels in the differential diagnosis of hirsute anovulatory women

Polycystic ovarian syndrome (PCO) occurs frequently in hirsute patients. A dissociated explosive response of LH to LHRH administration has been associated with the diagnosis of PCO. Twenty-four of 58 women seen because of hirsutism were found to have ovarian dysfunction based on clinical signs such as anovulation and irregular menstrual cycles. Plasma androgen levels were elevated in the patient group. The LHRH test (200 micrograms, iv) was applied to the 24 patients and compared with 13 normal ovulatory controls. Serum FSH levels before and after the LHRH test were normal in all patients. Two patterns of LH response to LHRH stimulation were observed: an explosive response in 17 patients (delta LH: 39.4 +/- 21.8 IU/l, control group: 7.35 +/- 4.4 IU/l, P < 0.01) and a normal response in 7 patients (delta LH: 7.53 +/- 2.41 IU/l). There was a significant correlation (r = 0.63, P < 0.05) between basal LH levels and LH response to LHRH. Sensitivity and specificity calculated for basal LH levels higher than 6.0 IU/l, considering the LHRH test as reference, were 58 per cent and 85 per cent , respectively. The positive predictive value measuring the possibility of LH higher than 6.0 IU/l to be from a patient with PCO (explosive response to LHRH) was 92 per cent . These data suggest that, in hirsute anovulatory patients, basal LH levels may be a good predictor in the diagnosis of polycystic ovarian syndrome