Circulating activated suppressor T lymphocytes in aplastic anemia

We studied the mechanism of hematopoietic suppression in aplastic anemia by means of two-color flow microfluorometric analysis of lymphocyte subpopulations and correlated the results with the occurrence in vitro of hematopoietic suppression and interferon production. In 12 patients with aplastic anemia a striking increase was observed in a population of "activated" suppressor T lymphocytes, which were defined by binding of both anti-Leu-2 and anti-HLA-DR monoclonal antibodies (patients with aplastic anemia, 6.8 +/- 3.2 per cent [mean +/- S.D.]; normal subjects, 1.7 +/- 1.3; patients given multiple transfusions, 2.5 +/- 1.7). Tac antigen expression, another surface marker of lymphocyte activation, was increased on suppressor lymphocytes in all five patients examined (patients with aplastic anemia, 31 +/- 17 per cent; normal subjects, 0.7 +/- 0.24; patients given multiple transfusions, 2.3 +/- 1.2). When Tac+ and Tac- cells were separated in a cell sorter, only Tac+ cells produced interferon. When lymphocytes of patients with aplastic anemia were cocultured with normal bone marrow, only the Tac+ cell fraction showed hematopoietic suppressor activity. In one patient, in vitro elimination of suppressor lymphocytes by use of OKT8 antibody abolished spontaneous interferon production by bone-marrow cells. These results suggest that activated suppressor lymphocytes producing interferon have a role in the pathogenesis of bone-marrow failure, and indicate the usefulness of defined lymphokine and phenotypic markers in the study of aplastic anemia.     

Guinea-pig peritoneal macrophage receptor for IgG--II. Purification of the receptor and its partial characterization

The Fc gamma receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of 125I-labeled macrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the 125I-labeled receptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 42-82%. Interactions of the purified receptor with IgG-Sepharose were equally well inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab')2 fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4 degrees C without a significant loss of the specific binding activity. Analysis of the receptor by SDS-polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.     

Reactivity of different LH and FSH standards and preparations in the world health organization matched reagents for enzyme-linked immunoassays of gonadotrophins

The immunoreactivity of various LH and FSH calibration standards and recombinant preparations in the enzyme-linked immunoassay (EIA) systems for gonadotrophins developed for the Special Programme of Research in Human Reproduction of the World Health Organization (WHO) were compared. The preparations tested included three LH and two FSH pituitary standards (calibrated against LH 80/552 and 68/40 and FSH 78/549 respectively) provided with the EIA or radioimmunoassay WHO matched reagent kits, the pituitary preparation LER-907, and recombinant human LH (rhLH) and FSH (rhFSH). Simultaneous curve fitting of the EIA dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards and LER-907; in contrast, no parallelism was found between the curves of rhLH and the pituitary-derived LH standards. No significant differences were found among the slopes of the curves elicited by the pituitary and recombinant FSH preparations. Each LH preparation exhibited a high degree of charge heterogeneity. Considerable variations in charge isoform distribution among the WHO LH standards, rhLH and LER-907 were also evident. In contrast, the FSH preparations were less heterogeneous and exhibited minor differences in charge distribution. Despite the existing differences in charge isoform distribution, all the pituitary-derived preparations as well as rhFSH seem appropriate for using as calibration standards in this particular EIA system.     

Investigation for a more virulent variant among the c:2b:P1.2,5 Spanish meningococcal epidemic strains by molecular epidemiology

A rise in the incidence of meningococcal disease has occurred in Spain in recent years, especially in some regions in the north-west of the country. Most cases have been caused by meningococci characterised as Neisseria meningitidis C:2b:P1.2,5. A total of 107 C:2b:P1.2,5 meningococcal isolates (60 from patients and 47 from carriers) and 12 isolates showing related antigenic combinations (C:2b:NST, C:2b:P1.2, C:2b:P1.5, C:NT:P1.2,5) was analysed by pulsed-field gel electrophoresis to determine the genetic variability of the epidemic and related strains. Endonucleases BglII and NheI were used to cut chromosomal DNA. When BglII was used, most of the C:2b:P1.2,5 isolates showed the same pulsotype regardless of whether they were from clinical cases or carriers. Isolates showing the principal profile after digestion with endonuclease BglII were analysed with NheI. Four pulsotypes were identified, of which two were found in only one isolate each. The major profiles (1 and 2) showed differential distribution among clinical and carrier isolates; pulsotype 1 was the most frequent among clinical isolates. However, the proportions of isolates showing profiles 1 and 2 were similar among carrier isolates. This could indicate that there are two variants of the C:2b:P1.2,5 strain with differing pathogenicity.     

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1–5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCα to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

Allergen-dependent CD14 modulation and apoptosis in monocytes from allergic patients

BACKGROUND: CD14 is a most important monocyte surface molecule. Recently, it has been reported that there is an important relationship between CD14 and immunoglobulin E, and that regulation of CD14 expression is an effector mechanism mediating apoptosis of monocytes. OBJECTIVE: The present study was designed to determine whether specific allergens were able to modulate CD14 expression and apoptosis by monocytes from allergic patients or whether specific immunotherapy (IT) might affect these processes. METHODS: One group of adult allergic asthmatic patients had received IT for the previous 3 years. Another similar group was not treated with IT. We challenged peripheral blood monocytes from both groups of asthmatic patients in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. The cells were also challenged with allergen to which the patients were not sensitive. Monocytes from normal subjects were also challenged with allergens. Expression of CD14 on the monocyte surface was analyzed by flow cytometry, and soluble CD14 (sCD14) in culture supernatant by enzyme-linked immunosorbent assay. The three groups of subjects were challenged with allergens, and apoptosis was analyzed by flow cytometry. RESULTS: When monocytes from non-IT-treated asthmatic patients were cultivated with the allergens to which the patients were sensitive, a significant up-regulation on the monocyte surface was observed compared with results from the healthy group (P < 0.003) and from the IT asthmatic group (P < 0.003). A significantly higher sCD14 level was observed in the culture supernatant of the monocytes from the IT asthmatic group were observed compared with those from the healthy group (P < 0.001) and those from the non-IT asthmatic group (P < 0.001). A significantly higher apoptosis level was observed in monocytes from the IT asthmatic group compared with those from the healthy group (P < 0.001) and those from the non-IT asthmatic group (<0.001). CONCLUSIONS: We present evidence that the expression of CD14 on the surface of monocytes and the apoptosis of the same cells can be modulated by an allergen-dependent mechanism. These processes can be affected by IT.