Studies on the specificities of two IgM lambda cold agglutinins

The reactions of two monoclonal IgM lambda cold agglutinins, Sch and Sher, have been studied in detail with human and animal erythrocyte antigens. Although they were unusual in having lambda and not kappa polypeptide chains, they could be assigned to the anti I and anti-Pr₁ groups of cold agglutinins.

The findings with serum Sher indicated that the Pr₁ antigen may be more complex than previously thought. The occurrence of unexpectedly large numbers of specificities among monoclonal anti-I, anti-i and anti-Pr antibodies is discussed and it is suggested that each monoclonal antibody may recognize only a limited portion of a complex red cell antigen.

     

Evaluation of the immunosuppressive effects of cyclophosphamide in patients with multiple sclerosis.

In a group of eight patients suffering from clinically definite multiple sclerosis, we studied the effects of treatment with cyclophosphamide on the immune reactivity in vitro and in vivo. The results are compared with those obtained in a control group consisting of eight patients who received no drug therapy and who were matched with the former group for age, sex and severity of disease. The results indicate that therapy with cyclophosphamide at a mean dose of 100 mg/day induces a profound lymphocytopenia in peripheral blood involving both T and B cells. Serum levels of immunoglobulins as well as primary and secondary antibody responses were depressed. In tests with standardized cell numbers, proliferative responses of lymphocytes in vitro and cytotoxic T cell function remained normal, whereas K and NK cell activities were diminished. Secondary cellular immune responses in vivo remained intact; however, the primary cellular immune response in vivo was markedly depressed. From these data, it is concluded that therapy with cyclophosphamide in man mainly affects humoral immune functions, but also cellular immunity, although to a lesser extent.     

Stenting for restenotic lesions with the BARD XT stent

BACKGROUND: Conventional PTCA for the treatment of restenotic lesions is associated with a high rate of recurrence (30-50%). Primary stenting decreases the restenosis rate at long-term follow-up. METHODS: One-hundred consecutive patients with restenosis received a Bard XT stent. Follow-up angiography was performed after 6 months. Angiograms were compared by means of computed quantitative analysis. RESULTS: The mean pretreatment reference diameter was 2.88 +/- 0.51 mm. The mean minimal luminal diameter (MLD) increased from 1.09 +/- 0.57 mm to 2.70 +/- 0.44 mm. The percent diameter stenosis decreased from 66 +/- 13% to 15 +/- 10%. The procedural success rate was 99%. At 6 month follow-up repeat angiography was performed in 86 patients. The mean MLD was 1.74 +/- 0.67 mm with a mean diameter stenosis of 41 +/- 20%. Residual anginal complaints were reported in 29% of patients. In-stent restenosis (defined as diameter stenosis of more than 50%) occurred in 18% of the patients. CONCLUSION: Placement of the Bard XT stent in restenotic lesions is feasible, has an excellent short term outcome and yields a favorable result at 6 month follow-up angiography.     

Haemodynamic effects of patent foramen ovale and atrial septal defect closure: a comparison during percutaneous shunt closure

Objectives: We investigated the haemodynamic effect of percutaneous closure of an intra‐atrial shunt, using non‐invasive finger pressure measurements. Background: Percutaneous closure of both patent foramen ovale (PFO) and atrial septal defect (ASD) is widely practised. Currently no data are available on short‐term haemodynamic changes induced by closure. Methods: Twenty‐five consecutive patients (mean age 49 ± 17 years, 10 men) who underwent a percutaneous closure of a PFO (n = 15) or ASD (n = 10) were included in this study. During the procedure blood pressure and heart rate (HR) were monitored continuously with a Finometer^®. Changes in systolic, mean, and diastolic pressure, stroke volume (SV), cardiac output (CO) and total peripheral resistance (TPR) were computed from the pressure registrations using Modelflow^® methodology. Results: Baseline characteristics were similar for the PFO and ASD patients. After PFO closure none of the haemodynamic parameters changed significantly. After ASD closure the systolic, mean, and diastolic pressures increased 7·1 ± 5·4 (P = 0·003), 3·8 ± 3·5 (P = 0·007) and 2·0 ± 3·0 mmHg (P = ns) respectively. HR decreased 5·1 ± 5·3 beats per minute (P = 0·01). SV, CO and TPR increased 8·5 ± 6·4 ml (13·5%; P = 0·002), 0·21 ± 0·45 l min^?1 (5·6%; P = ns) and 0·02 ± 0·14 dynes (4·1%; P = ns) respectively. The changes in SV differ between the PFO and ASD patients (P = 0·009). Conclusions: Using non‐invasive finger pressure measurements, we found that SV, mean and systolic blood pressure increased immediately after percutaneous closure of an ASD in adults, whereas the percutaneous PFO closure had no effect on haemodynamic characteristics.     

An Optimized CFSE‐based T‐cell Suppression Assay to Evaluate the Suppressive Capacity of Regulatory T‐Cells Induced by Human Tolerogenic Dendritic Cells

In autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are considered to have a strong potential as cellular therapy for these diseases. One of the mechanisms of immune suppression mediated by tolerogenic DC is the induction of regulatory T‐cells (Treg). Consequently, the efficacy of such DC to induce Treg will reflect their tolerogenic capacity. Because no specific markers have been described for human induced (i)Treg yet, the Treg can only be appreciated by functionality. Therefore, we have optimized an in vitro suppression assay to screen for human DC‐induced‐Treg activity. IL‐10‐generated tolerogenic DC were used to induce Treg that were previously shown to effectively suppress the proliferation of responder T‐cells stimulated with allogeneic mature DC (mDC). Our results show that the suppressive capacity of IL‐10 DC‐induced Treg measured in the suppression assay increases with the iTreg dose and decreases with higher numbers of antigen‐presenting cells (APC) as T‐cell stimulation. Lowering the ratio between responder T‐cells and stimulator mDC present in the coculture clearly improved the read‐out of the suppression assay. Furthermore, mDC‐primed T‐cells in the suppression assay were shown to be an essential control condition. In conclusion, we recommend titrations of both APC and iTreg in the suppression assay and to include a negative control condition with T‐cells primed by mDC, to distinguish specific and functional suppression by iTreg from possible generalized suppressive activity.     

Reduced Cbl phosphorylation and degradation of the zeta-chain of the T-cell receptor/CD3 complex in T cells with low Lck levels

T cells with short interfering RNA-mediated Lck-knockdown (kd) display paradoxical hyper-responsiveness upon TCR ligation. We have previously reported a possible mechanism for T-cell activation in cells with low levels of Lck depending on Grb2-SOS1 recruitment to the zeta-chain of TCR/CD3 (Methi et al., Eur. J. Immunol. 2007, 37: 2539-2548). Here, we show that short interfering RNA-mediated targeting of Lck caused a dramatic reduction in c-Cbl phosphorylation and a general reduction in protein ubiquitination after TCR stimulation. Specifically, this resulted in reduced ubiquitination of the zeta-chain, yet internalization of TCR/CD3 appeared to be normal after receptor engagement. However, zeta-chain levels were elevated in Lck-kd cells, and confocal microscopy revealed reduced colocalization of CD3-containing vesicles with endosomal and lysosomal compartments. We hypothesize that prolonged stability of internalized T-cell receptor complex may result in extended signaling in T cells with low Lck levels.     

Short‐term cytokine stimulation reveals regulatory T cells with down‐regulated Foxp3 expression in human peripheral blood

The identification of regulatory T cells (Treg cells) in human peripheral blood is an important tool in diagnosis, research, and therapeutic intervention. As compared to lymphoid tissues, the frequencies of circulating Treg cells identified as CD4⁺CD25⁺Foxp3⁺ are, however, low. We here show that many of these cells remain undetected due to transient down regulation of Foxp3, which rapidly decays in the absence of cytokine‐mediated STAT5 signals. Short‐term incubation of PBMCs or isolated CD4⁺ T cells, but not of lymph node cells, with IL‐2, ‐7, or ‐15 more than doubles the frequency of Foxp3⁺CD25⁺ among CD4⁺ T cells detectable by flow cytometry. This increase is not due to cell division but to upregulation of both proteins. At the same time, the uncovered Treg cells up‐regulate CD25 and down‐regulate CD127, making them accessible to viable cell sorting. “Latent” Treg cells have a demethylated FOXP3 TSDR sequence, are enriched in naïve, non‐cycling cells, and are functional. The confirmation of our findings in RA and SLE patients shows the feasibility of uncovering latent Treg cells for immune monitoring in clinical settings. Finally, our results suggest that unmasking of latent Treg cells contributes to the increase in circulating CD4⁺CD25⁺Foxp3⁺ cells reported in IL‐2 treated patients.