Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.     

CD7+, CD4-, CD8- acute leukemia: a syndrome of malignant pluripotent lymphohematopoietic cells

Following our initial observation of in vivo conversion of CD7+, CD4-, CD8- acute lymphoblastic leukemia (ALL) cells from lymphoid to myeloid lineages (Proc Natl Acad Sci (USA) 81:253, 1984) we have studied eight additional cases of ALL with this leukemic cell phenotype. The CD7+, CD4-, CD8- phenotype was associated with a distinct clinical entity with those affected predominantly male (either less than 35 years or greater than 65 years of age), with frequent mediastinal and/or thymic masses, skin and CNS disease, high peripheral WBC counts, and bone marrow blasts that were morphologically L1 or not ascribable to a specific lineage. These patients did not respond to conventional chemotherapeutic regimens for either acute lymphoid or myeloid leukemias. No common karyotype or T-cell gene rearrangement pattern could be defined. Importantly, seven of eight patient's leukemic cells studied were capable of multilineage (myeloid, erythroid, monocytoid, megakaryocytoid, and lymphoid) differentiation in vitro. Data is presented suggesting that CD7+, CD4-, CD8- leukemias, in many instances, are leukemias of immature hematopoietic cells. The development of novel therapeutic approaches to this form of leukemia will be necessary to alter its poor prognosis.     

Biological effects of recombinant human granulocyte colony-stimulating factor in patients with untreated acute myeloid leukemia

Hematopoietic growth factors are being administered to patients with acute myeloid leukemia (AML) both to shorten the duration of chemotherapy-induced neutropenia and in an attempt to increase cytotoxicity of cell cycle-specific agents. However, limited information is available concerning the effects of growth factors in AML patients. To examine the in vivo effects of recombinant human granulocyte colony-stimulating factor (G-CSF) on AML cells, laboratory studies were performed before and after a 72-hour intravenous infusion of G-CSF (10 micrograms/kg/d) administered to 28 untreated AML patients. Twenty-seven patients (96%) showed increases in at least one of the following parameters after G-CSF: blood blasts, bone marrow (BM) blasts, leukemia cells in S phase or interphase cells with leukemia- specific markers shown by fluorescence in situ hybridization. The median paired change in absolute blast count was +2.7 x 10(9)/L (P = .0001) after G-CSF, as compared with 0.0 during the 72 hours before initiation of G-CSF. The median percentage of BM leukemia cells in S phase increased from 6.0% to 10.7% after G-CSF (median change, %5.9%; P = .009). Interphase BM cells with trisomy 8 or monosomy 7 increased in 6 of 6 patients with these abnormalities (P = .02) with a median percent increase of 47%. Blood neutrophil counts also increased during G-CSF (median paired change, +2.8 x 10(9)/L; P < .0001). Trisomy 8 or monosomy 7 was shown by fluorescence in situ hybridization in post-G- CSF blood neutrophils from 4 of 6 patients but was also present in neutrophils before G-CSF. We conclude that the percentage of leukemia cells in S phase increases and that leukemia cell populations undergo expansion during short-term administration of G-CSF in almost all AML patients.     

Soluble interleukin 2 receptors in sera of Japanese patients with adult T cell leukemia mark activity of disease

Serum concentrations of soluble interleukin 2 receptors (sIL 2R) were measured by an enzyme-linked immunosorbent assay (ELISA) in 30 patients with adult T cell leukemia (ATL), in 9 patients with other hematopoietic malignancies, and in 17 asymptomatic individuals seropositive for human T cell leukemia virus type I (HTLV-I). Sixty HTLV-I seronegative, age-matched controls showed a normal range of form 63.2 to 480.8 U/mL. All asymptomatic carriers of HTLV-I had sIL 2R in their sera within the normal range. sIL 2R in sera was not related to the anti-HTLV-I antibody titer. Eleven patients with acute ATL, a clinical phenotype with median survival rate of 4.4 months, had markedly elevated sIL 2R (11,100 to 99,000 U/mL), but eight patients with smoldering ATL had low sIL 2R values (less than 480.8 U/mL) comparable to controls. Eleven patients with chronic ATL had intermediate elevated levels of sIL 2R (480.8 to 37,300.0 U/mL). Serum levels of sIL 2R correlated with the number of ATL cells (r = 0.812) and CD25-positive cells (r = 0.725) circulating in the peripheral blood. Longitudinal studies performed in four patients with ATL showed significant correlation between serum concentration of sIL 2R and activity of the malignancy. These findings suggest that the level of sIL 2R in serum indicated tumor load and, possibly, prognosis.     

The effects of dietary omega 3 fatty acids on platelet composition and function in man: a prospective, controlled study

The rarity of atherosclerotic vascular disease and a mild bruising tendency in Greenland Eskimos has been linked to their ingestion of omega 3 fatty acids contained in foods obtained from the sea. Previous studies have shown that feeding salmon oil to normal volunteers resulted in reductions of plasma cholesterol and triglycerides. We wished to learn whether salmon oil feeding would result in the incorporation of omega 3 fatty acids into platelets and whether platelet function or platelet-vessel interaction would be altered. Diets containing salmon oils led to the incorporation of eicosapentaenoic acid (C20:5 omega 3) into platelets (6.1%) with a reduction in arachidonic acid (C20:4 omega 6). The ratio of C20:5/C20:4 increased from 0.0045 on the control diet to 0.3 on the salmon diet. Bleeding times were prolonged (from 6.75 to 10 min, p less than 0.005), platelet retention on glass beads was mildly reduced (from 89% to 78%, p less than 0.0005), and platelet aggregation in response to dilute concentrations of ADP was inhibited in the subjects ingesting the salmon oil. We conclude that in normal subjects dietary omega 2 fatty acids derived from salmon oil are incorporated into platelet phospholipids and that these changes are accompanied by alterations in bleeding time and platelet function.     

The Effect of Argon and Nitrogen Ion Implantation on Nickel-Titanium Rotary Instruments

IntroductionThis qualitative study investigated the effect of N₂⁺ and Ar⁺ ion implantation on morphologic alterations and fatigue resistance in Pro Taper S1 NiTi (Dentsply-Maillefer, Ballaigues, Switzerland) rotary instruments.MethodsInstruments were divided into three groups: N₂⁺ implanted, Ar⁺ implanted, and unmodified control group. All instruments were used to prepare five curved canals in epoxy resin blocks with brushing motion. The instruments were examined in a scanning electron microscope (SEM) before use, after first use, and after the fifth use. A more demanding cyclic fatigue test was undertaken, submitting the instruments to 15-second periods of continuous rotation inside the curved canals without a brushing motion. Crack formation was analyzed with the SEM, and the number of 15-second periods required to fracture each instrument was recorded.ResultsNo significant morphologic alterations were observed in the instruments after the preparation of five canals. Crack density was similar in all groups. In the subsequent cyclic fatigue test, instruments implanted with nitrogen performed worse than those implanted with argon and the control group. Fracture faces show differences in the fracture modes.ConclusionsAr⁺ implantation improved the performance of S1 files moderately, whereas nitrogen ion–implanted files performed worse in the fatigue test. A reduction in file performance seems to be caused by nitrogen diffusion in the grain boundaries, instead of the desired improvement caused by titanium nitride formation.     

Cerebrospinal fluid cytokine levels in migraine, tension-type headache and cervicogenic headache

Cytokines have been measured in cerebrospinal fluid (CSF) from headache patients [infrequent episodic tension-type headache (TTH) and migraine with or without aura, all during attack, and cervicogenic headache] and compared with levels in pain-free individuals. Both proinflammatory [interleukin (IL)-1β, tumour necrosis factor-α and monocyte chemoattractant protein-1 (MCP-1)] and anti-inflammatory cytokines [IL-1 receptor antagonist (IL-1ra), IL-4, IL-10 and transforming growth factor-β1 (TGF-β1)] were included. There were significant group differences in IL-1ra, TGF-β1 and MCP-1 in episodic TTH and migraine compared with controls, and a significant difference in MCP-1 between cervicogenic headache and migraine with aura. Intrathecal MCP-1 correlated with IL-1ra, IL-10 and TGF-β1 in episodic TTH, and MCP-1 with IL-10 in migraine with aura. Cytokine increases were modest compared with those often accompanying serious neurological conditions, and may represent a mild response to pain. We believe this to be the first comparative study of CSF cytokine levels in connection with headache.     

Depressive symptoms in patients with chronic hepatitis C are correlated with elevated plasma levels of interleukin-1beta and tumor necrosis factor-alpha

Studies suggest that cytokines have a role in the biology of depression. In this study, we evaluated depression and cytokine levels in patients with and without chronic hepatitis C (HCV) to better assess how chronic infection alters cytokines levels and may contribute to depressive symptomotology. Twenty-three adults with (n=16) and without (n=7) HCV were recruited through the Portland VA Medical Center. Research participants were excluded for current substance abuse, psychotic disorder, liver cirrhosis, or interferon (IFN) therapy. Participants completed the Beck Depression Inventory-II (BDI-II) and a blood draw to evaluate plasma cytokine levels [i.e., interleukin (IL)-1beta, IL-10 and tumor necrosis factor (TNF)-alpha]. t-Tests were performed to compare cytokine levels in patients with or without HCV. HCV patients showed higher TNF-alpha values compared to patients without HCV (group means=7.94 vs. 3.41pg/mL, respectively, p=0.047). There were no significant differences between the groups for the other cytokines assessed. In patients with HCV, TNF-alpha and IL-1beta levels (but not IL-10) were correlated with BDI-II scores [r=0.594, p=0.020 and r=0.489, p=0.055 (trend), respectively]. Taken together, these results show an association between severity of depressive symptoms and expression of pro-inflammatory cytokines in patients with HCV. Future studies should investigate how inflammatory mediators play a role in the expression of specific depressive symptoms in patients with chronic infection. Patients with HCV represent an interesting model to examine this relationship.     

Costimulation of Th17 cells: adding fuel or putting out the fire in the inflamed gut?

Inflammatory bowel disease, typified by Crohn’s disease and ulcerative colitis, is a common disorder characterized by recurrent and serious inflammation of the gastrointestinal tract. It is well documented that T cells play a pivotal role in the development of inflammatory bowel disease. Th17 cells are a unique T cell subpopulation implicated in inflammatory bowel disease and many other autoimmune/inflammatory diseases. However, the regulatory mechanism of Th17 activation and proliferation has not been defined completely. Recent studies have shown that the ligation of several costimulatory receptor–ligand pairs contributes to the activation, differentiation, and proliferation of T lymphocytes including the Th17 subset. In this review, we will discuss the emerging evidence on the role of Th17 cells in inflammatory bowel disease pathogenesis as well as the effect of costimulatory molecules on Th17 development and consider if the need for such costimulation of T lymphocytes provides a target for the development of novel therapeutic strategy.