E. Marion Schneider

To characterize the immune modulatory effects of 5-HT3 receptor antagonist treatment in patients with fibromyalgia, autoimmune disorders, and chronic pain. Multiplex-assisted cytokine measurements were performed before and during treatment. Whole blood stimulation with TNF-alpha was carried out to determine the proinflammatory response induced by exogenous TNF-alpha. Five of nine patients clinically responded to treatment, and two had a moderate response. All patients had significantly elevated levels of T-H1 cytokines more prominent than TNF-alpha, IL-1beta, and IL-6. Treatment resulted in transient effects on peripheral monocyte counts in all but one patient, a plasma IL-1beta increase in two responder patients, and decreased T-H1 cytokines in two responder patients. Ex vivo TNF-alpha stimulation was transiently reconstituted in three responder patients to a significant level. Three patients showed a marginal reconstitutive response. 5-HT3 receptor blockade transiently affects monocyte tissue infiltration, modulates T-H1 cytokines in clinical responders as well as MIP-1beta in moderate responders, and transiently affects the ex vivo response to exogenous TNF-alpha.

Abstract: Microvascular endothelial cells (mECs) circulate at higher numbers in patients with severe sepsis and hemophagocytic syndromes. Although these blood mECs might stem from damaged microvasculature, they are perfectly viable and lead to the establishment of cell lines. Such mECs were cultured in low‐dose human serum pools (0.5%) and MEM‐alpha medium. Antigenic profiling revealed the expression of CD36, factor VIIIa, CD95‐ligand, and CD44, but also CD146. We studied the antioxidative effect of the hematopoietic growth factor G‐CSF1 after in vitro stimulation with LPS from E. coli 0111:B4; the growth factor appeared to exhibit a protective effect on organ function in patients with SIRS. mECs were stimulated with 1 μg/mL of LPS for 24 h and 48 h with and without G‐CSF (3×10³ U/mL) preincubation. After 24 h, supernatants of the stimulated mEC were tested for IL‐8 by ELISA, and cells were tested for hemoxygenase‐1 (HO‐1, Hsp32) by immunohistochemistry and flow cytometry using OSA110 (mAb, Stressgene). Stimulation with LPS upregulated IL‐8 by a factor of 2 to 10 in mEC. Preincubation with G‐CSF markedly downregulated the LPS‐induced IL‐8 secretion (20–50%), but IL‐6 production was not affected. Upon 48 h of LPS stimulation, mECs developed massive signs of apoptosis and concomitant caspase 3 activation. Caspase 3 activity induced by LPS (24 h) or by staurosporin (6 h) was found to be dramatically downregulated by the G‐CSF preincubation protocol.

In 65 patients with hemophagocytic lymphohistiocytosis (HLH), we found an as yet undescribed heterogeneity of defects in cellular cytotoxicity when assay conditions were modified by the incubation time, the presence of mitogen, or interleukin-2 (IL-2). The standard 4-hour natural killer (NK) test against K562 targets was negative in all patients. In patients deficient in type 1 (n = 21), type 2 (n = 5), and type 4 (n = 8) HLH, negative NK function could be reconstituted by mitogen, by IL-2, or by prolongation of the incubation time (16 hours), respectively. Most patients (n = 31) displayed the type 3 defect, defined by a lack of any cellular cytotoxicity independent of assay variations. The characteristic hypercytokinemia also concerned counterregulatory cytokines, such as proinflammatory interferon-γ (IFN-γ), simultaneously elevated with suppressive IL-10 in 38% of types 1–, 2–, and 4–deficient patients and in 71% of type 3–deficient patients. Elevated IFN-γ alone correlated with high liver enzymes, but sCD95-ligand and sCD25 did not—though these markers were expected to indicate the extent of histiocytic organ infiltration. Outcome analysis revealed more deaths in patients with type 3 deficiency (P = .017). Molecular defects were associated with homozygously mutated perforin only in 4 patients, but other type 3 patients expressed normal transcripts of effector molecules for target-cell apoptosis, including perforin and granzyme family members, as demonstrated by RNase protection analysis. Thus, target-cell recognition or differentiation defects are likely to explain this severe phenotype in HLH. Hyperactive phagocytes combined with NK defects may imply defects on the level of the antigen-presenting cell.