Brain-Spleen Inflammatory Coupling: A Literature Review.

Recent evidence suggests a link between brain injury and the autonomic release of pro-inflammatory cytokines by resident macrophages in the spleen. This phenomenon, termed "brain-spleen inflammatory coupling," has garnered attention from scientific and medical communities interested in developing novel treatments for traumatic brain injury (TBI). Cholinergic stimulation of the α7-subunit nicotinic acetylcholine receptor (α7NAchR) on splenic macrophages has been shown to inhibit their release of pro-inflammatory cytokines. This inhibition, mediated by the parasympathetic nervous system, has been shown to improve outcomes in animal models of sepsis, stroke, and TBI. As evidence of a beneficial role of splenic inhibition grows, new treatment strategies might be applied to many medical conditions involving neuroinflammation, a process that contributes to further neurological deterioration.

Optimized culture conditions for the generation of dendritic cells from peripheral blood monocytes.

BACKGROUND AND OBJECTIVES: Dendritic cells (DCs) are promising adjuvants for clinical immunotherapy, but they are scantily distributed. Therefore, numerous in vitro methods have been developed to expand these cells while maintaining their normal functions. Current culture systems generally require the use of fetal bovine serum (FBS)-supplemented media in order to attain DCs with high immunostimulatory activity. However, the presence of exogenous animal proteins sets limits for their use in clinical trials. The purpose of this study was to establish a simple, efficient and FBS-free method for the generation of human DCs for clinical application. MATERIALS AND METHODS: We compared monocyte-derived DCs generated in a standard FBS-supplemented medium vs. DCs generated in an autologous plasma (AutoPl)-supplemented medium, with regard to their yield, function and longevity. Peripheral blood monocytes were isolated from buffy coats by two consecutive 2-h adherence steps in tissue culture flasks. The adherent cells were differentiated into DCs within 2 weeks by adding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), c-kit ligand and tumour necrosis factor-alpha (TNF-alpha). Every 2-3 days, the cells in suspension were analysed for their immunophenotype and apoptosis rate by flow cytometry. Their function was demonstrated by their allostimulatory and migratory capacity, as well as by their proteolytic activity. RESULTS: We show that more than 30 x 10(6) DCs can be achieved per unit of buffy coat using either AutoPl- or FBS-supplemented media. The purity of the DCs was 53.4% and 65% (P > 0.05) in AutoPl- and FBS-based medium, respectively. DCs grown in AutoPl media showed a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1aneg phenotype, while FBS-generated DCs exhibited a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1ahigh phenotype. The apoptosis rate in both culture conditions increased from 10% to 25% over 1 week. AutoPl-generated DCs were shown to be equally strong stimulators for proliferation of allogeneic T lymphocytes as FBS-generated DCs. In addition, the capacity to migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha) and stromal-cell-derived factor 1alpha (SDF-1alpha) was similar in both groups, whereas the response to MIP-3beta was reduced in AutoPl-derived cells. Zymography analysis of supernatants from 5-day-old cultures demonstrated that AutoPl-generated DCs produced higher amounts of matrix metalloproteinases, suggesting that they have an enhanced capability to traffic through peripheral tissues. CONCLUSIONS: Our findings indicate that plastic-adherent peripheral blood cells, when cultured with GM-CSF, IL-4, c-kit-ligand and TNF-alpha in autologous human plasma-supplemented media, are a potent source of functional DCS that may be of value for human therapy.

Transmission of human T-cell lymphotropic virus type 1 tax to rabbits by tax-only-positive human cells.

The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40tax in their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40(tax). Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1 tax sequences and antibodies to p40(tax), while they lacked sequences and antibodies related to gag, pol, or env. Thus, it seemed important to determine whether the "tax-only" state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegative tax-only-positive MF patients or from healthy tax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits became tax sequence positive. These observations suggest that HTLV-1 tax can be transmitted by tax-only-positive mononuclear cells.

Structural abnormalities in the G-CSF receptor in severe congenital neutropenia.

Structural abnormalities in the cytoplasmic region of the G-CSF receptor (G-CSF-R) or defects in signal transduction pathways triggered by the G-CSF-R or both have been implicated in the development of neutropenia and increased prediposition to leukemia in patients with severe congenital neutropenia (SCN). To assess the structural integrity of the G-CSF-R in SCN patients, the transmembrane and cytoplasmic regions of the G-CSF-R from 5 SCN patients were cloned and sequenced. DNA mutations (point, deletion, frame-shift, and silent) were observed in 3 patients. In 2 of these, the DNA mutations resulted in altered G-CSF-R protein sequences, including additions of novel C-terminal sequences. Three of the 5 mutant receptor clones lacked 115-121 amino acids in the cytoplasmic region, and 2 showed complete loss of the transmembrane and cytoplasmic regions. Neutrophils from 1 patient expressing these mutant receptors showed normal binding of radiolabeled G-CSF. G-CSF-R in 2 other patients with SCN showed no mutations. Our results indicate that structural abnormalities in the G-CSF-R may be present in some SCN patients. They may not affect the binding of G-CSF to the receptor but may contribute to the pathogenesis of SCN through impaired signal transduction pathways of the mutant G-CSF-R.

Apyrase, ascorbic acid and aprotinin ameliorate the storage lesion in pelleted platelet preparations.

To evaluate the effect of apyrase, ascorbic acid and aprotinin (AAA) in preventing platelet activation during storage, 12 sets of platelet concentrates (PCs), were treated with AAA and evaluated at days 1, 3, and 5 utilizing platelet functional and morphological assays. Platelets treated with AAA demonstrated significantly enhanced response to ADP-induced platelet aggregation, higher morphology scores, and evaluated ATP levels compared to control samples after 5 days of storage. Similarly, platelet specimens treated with AAA had significantly reduced PF4 secretion and P-selectin expression compared to controls. Finally, Western blots of aggregated platelets at day 5 demonstrated that AAA-treated PCs continue to express the platelet membrane GPIb whereas specimens from control PCs do not. These results show that PCs treated with AAA have reduced platelet activation and enhanced functional platelet activity.

Specificity analysis of murine monoclonal antibodies reactive with Tn, sialylated Tn, T, and monosialylated (2-->6) T antigens.

T, Tn, and sialyated Tn (sTn) are pancarcinoma antigens, and increased expression of these carbohydrate epitopes has been correlated with a poor prognosis in several epithelial malignancies. Ten murine monoclonal antibodies have been generated to these antigens, and compared by ELISA and immunohistochemistry to established mAbs reactive with these antigens. Nine mAbs (3 IgM and 6 IgG) reactive with synthetic T-human serum albumin (T-HSA) were produced after immunizing BALB/c mice with a synthetic T-keyhole limpet hemocyanin glycoconjugate (T-KLH). An additional IgM mAb (145.22) was produced in mice immunized with erythrocytes isolated from a patient with Tn syndrome. Three IgM and six IgG1 mAbs reactive with T-HSA did not react with natural T antigen present on desialyated glycophorin. All three IgM and several IgG1 mAbs, however, did react with LS-174T, a mucinous colon carcinoma cell line, 647V, a human bladder carcinoma cell line, and TA3Ha, a murine mammary carcinoma cell line as well as fresh frozen colon carcinomas. MAb 145.22 reacted with both natural and synthetic sources of sTn and Tn, as well as with LS-174T cells and mucin deposits in 10/11 colon carcinomas on fresh-frozen sections. MAb B72.3 reacted strongly with ovine submaxillary mucin (OSM) and sTn-HSA, while mAb CC49, a second-generation mAb to TAG-72 carcinoma mucin, reacted strongly with OSM, less strongly with desialyated OSM, and only weakly with sTn-HSA, suggesting that the epitope specificity for mAb CC49 is distinct from that of B72.3.

A novel technique for preparing improved buffy coat platelet concentrates.

We evaluated in vitro platelet function of platelet concentrates stored at 22 C for 5 days prepared either by the conventional pelleting procedure or platelet concentrates prepared from buffy coats by utilizing a novel bucket designed to support a suspended bag. For platelet concentrates from buffy coat, whole blood was centrifuged at 3,000 x g for 13 min, with all but 30cc of the cell poor plasma transferred to a satellite bag, followed by a second centrifugation at 170 x g for 5 min utilizing our novel centrifugation device. For pelleted platelets, whole blood was centrifuged at 2,000 x g for 3 min, platelet rich plasma removed, centrifuged, and the pellet resuspended in plasma. Leukocyte contamination in buffy coat platelet concentrates was reduced by 95% (p < 0.001) in comparison to pelleted platelets. Further, platelets from buffy coat platelet concentrates demonstrated significantly enhanced ADP-induced aggregation, increased recovery from hypotonic shock, higher morphology scores, and reduced GMP-140 expression in comparison to pelleted preparations. No differences in O2 consumption, CO2 production, pH and total ATP were observed between the two types of preparations at day 5 of storage. Our results indicate that platelet concentrates from buffy coat, prepared by a suspended storage bag centrifugation technique, are superior with respect to in vitro platelet function when compared to pelleted platelets.

A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia.

Patients with idiopathic, cyclic, and congenital neutropenia have recurrent severe bacterial infections. One hundred twenty-three patients with recurrent infections and severe chronic neutropenia (absolute neutrophil count < 0.5 x 10(9)/L) due to these diseases were enrolled in this multicenter phase III trial. They were randomized to either immediately beginning recombinant human granulocyte colony-stimulating factor (filgrastim) (3.45 to 11.50 micrograms/kg/d, subcutaneously) or entering a 4-month observation period followed by filgrastim administration. Blood neutrophil counts, bone marrow (BM) cell histology, and incidence and duration of infection-related events were monitored. Of the 123 patients enrolled, 120 received filgrastim. On therapy, 108 patients had a median absolute neutrophil count of > or = 1.5 x 10(9)/L. Examination of BM aspirates showed increased proportions of maturing neutrophils. Infection-related events were significantly decreased (P < .05) with approximately 50% reduction in the incidence and duration of infection-related events and almost 70% reduction in duration of antibiotic use. Asymptomatic splenic enlargement occurred frequently; adverse events frequently reported were bone pain, headache, and rash, which were generally mild and easily manageable. These data indicate that treatment of patients with severe chronic neutropenia with filgrastim results in a stimulation of BM production and maturation of neutrophils, an increase in circulating neutrophils, and a reduction in infection-related events.

O-linked oligosaccharides of glycophorins A and B in erythrocytes of two individuals with the Tn polyagglutinability syndrome.

Tn polyagglutinability syndrome is an acquired condition where erythrocytes express Tn neo-antigen and become susceptible to hemagglutination by the naturally occurring anti-Tn present in normal sera. Early studies had indicated that O-linked N-acetyl galactosamine was the sole serologic Tn determinant, but more recently O-linked NeuNAc alpha 2, 6GalNAc also has been implicated as a Tn antigen (sialosyl-Tn). However, none of these studies were performed on purified glycoproteins. In this report we examine oligosaccharides of glycophorins A and B purified from Tn erythrocytes of two affected individuals to establish how N- and O-linked saccharides differ from normal. Analysis of carbohydrate composition and treatment with N-glycanase showed that the Asn-linked unit of glycophorin A was not affected. O-linked oligosaccharides were obtained by beta-elimination in the presence of tritiated sodium borohydride. The reduced radiolabeled products were fractionated by Bio-Gel P-2 chromatography, and their structures were investigated by comparison with standards, by monosaccharide quantification, and by neuraminidases of known specificities. The results show that Tn glycophorins from both donors contain intact and truncated forms of trisaccharide and tetrasaccharide NeuNAc alpha 2,3Gal beta 1,3GalNAc and NeuNAc alpha 2,3Gal beta 1,3- (NeuNAc alpha 2,6)GalNAc usually present in glycophorins A and B. The truncated forms include the protein O-linked monosaccharide, GalNAc and disaccharide, NeuNAc alpha 2,6GalNAc (major isomer). The presence of intact glycans in the total population of Tn erythrocytes was confirmed by their susceptibility to T activation after treatment with neuraminidase. The proportion of the four species was not identical in glycophorins of these two donors but, in both, the truncated units predominated and the amount of the disaccharide was approximately one half of that of the monosaccharide. The data are consistent with alterations in UDPGal:GalNAc beta 1,3galactosyl transferase that may have multiple molecular origins and with induction of a specific GalNAc protein alpha 2,6 sialosyl transferase in Tn hematopoietic precursor cells. The molecular basis for these alterations awaits further study.

New effectors of human hemoglobin: structure and function.

We describe the actions of two new allosteric effectors of hemoglobin, 2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid (L35) and 2-[4-(3,4,5-trichlorophenylureido)phenoxy]-2-methylpropionic acid (L345). Each of them binds to two pairs of symmetry-related sites in the central cavity of human deoxyhemoglobin. One pair of sites overlaps with that occupied by bezafibrate [Perutz et al. (1986) J. Am. Chem. Soc. 108, 1064-1078]. The other sites are new, and the pair occupied by L35 is different from that occupied by L345. All the sites are at least 20 A from the site where organic phosphates are bound. L345 is by far the most potent allosteric effector of hemoglobin ever described. At a concentration of 0.1 mM, it raises the P50 of a suspension of red cells by 50%; at 0.2 mM it raises the P50 2.5-fold. At acid pH, it reduces Hill's coefficient to near unity and prevents complete oxygen saturation even under 1 atm of pure oxygen. In azidemethemoglobin at pH 6, it induces a transition to higher spin. These properties are reminiscent of those of teleost fish hemoglobins that exhibit a Root effect. The influence of L35 and L345 and that of organic phosphates on the oxygen affinity are additive, but they compete with chloride. L35 acts more weakly than L345, but can be made to induce the same effects as L345 alone by adding inositol hexaphosphate. Both compounds increase the alkaline and acid Bohr effects. They alter the bimolecular kinetics of CO recombination after a flash by increasing the slowly reacting fraction of hemoglobin in the T state at the expense of the fast-reacting fraction in the R state.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and investigation of effects of 2-[4- [[(arylamino)carbonyl]amino]phenoxy]-2-methylpropionic acids on the affinity of hemoglobin for oxygen: structure-activity relationships.

A series of 2-[4-[[[(substituted-phenyl)amino]carbonyl]amino]phenoxy]-2- methylpropionic acids and other substituted phenoxyacetic acids were synthesized and tested for their ability to reduce the affinity of hemoglobin for oxygen. 2-[4-[[[(3,4,5-trichlorophenyl)amino]carbonyl]amino]phenoxy]-2- methylpropionic acid was found to be the most potent compound known. Structure-activity relationships of the compounds synthesized are discussed.

LR16, a compound with potent effects on the oxygen affinity of hemoglobin, on blood cholesterol, and on low density lipoprotein.

2-[4-(3,4-Dichlorophenylureido)phenoxy]-2-methylpropionic acid, LR16, combines with two symmetrically related sites in the central cavity of deoxyhemoglobin, 20 A away from the binding site of 2,3-bisphosphoglycerate, and acts as an allosteric effector synergistic with 2,3-bisphosphoglycerate. LR16 (1 mM) raises P50, the partial pressure of oxygen needed to achieve half-saturation with oxygen of a hemolysate of human hemoglobin, about 50 times more strongly than 1 mM 2,3-bisphosphoglycerate. Oral administration of LR16 (at small doses that produced no ill effects) to rats that were fed a diet rich in cholesterol caused substantial reductions of total serum cholesterol and low density lipoprotein-cholesterol, while high density lipoprotein-cholesterol remained unchanged.

Specificity of oxygen radical scavengers and assessment of free radical scavenger efficiency using luminol enhanced chemiluminescence.

The selective scavenging capacities of 19 important oxygen radical scavengers were determined by adding them individually to each of the four oxy radical standards, (superoxide, hydroxy, alkoxy and hydroperoxy, and singlet O2), calculating the percent chemiluminescence inhibited, and extrapolating O2 equivalents neutralized from baseline. The sensitivity (0.01 nm/ml) and selectivity of this method not only allows identification of individual oxygen free radical species but also quantitates the efficiency of free radical scavengers.

The manual hexadimethrine bromide (Polybrene) test. Effects of serum proteins and practical applications.

Serum proteins affect the performance of the manual hexadimethrine bromide (Polybrene) test. Red cells suspended in the low-ionic medium adhere to the surface of test tubes when centrifuged. This adherence is prevented by serum components apoprotein B, fibronectin, von Willebrand factor, or serum diluted less than 75-fold, but not by serum albumin or immunoglobulin fractions. A high concentration of serum reduces the sensitivity of the test and destabilizes antibody-dependent aggregation. Disaggregation time (DT), defined as the time required for antibody-dependent aggregates to disperse, was used as an indicator of agglutination stability. DT was often shortened when antibodies were in native serums, but could be prolonged if serums were diluted with 0.85 percent saline. Other factors that influenced DT included antibody concentration, temperature, specificity, and sources of antibodies within specificities. When antibodies were present in a mixture, they produced various DTs related to individual specificities. Determination of these differences allowed the successful identification of multiple antibodies. The procedure, called differential disaggregation time, was also used for typing red cells coated by autoantibodies. Additional modifications improved the performance of the antiglobulin phase.

Autoimmune neutropenia of infancy.

Neutrophil antibodies were demonstrated in 119 of 121 infants and young children with chronic neutropenia, establishing the diagnosis of autoimmune neutropenia of infancy. The median age at diagnosis was 8 months (range 3 to 30 months), and the female/male ratio was 6:4. Autoimmune neutropenia of infancy was manifested by recurrent fever and infection. All patients had selective neutropenia (absolute neutrophil count 0 to 500), and many had monocytosis. Fifteen of 16 patients tested failed to respond to epinephrine and hydrocortisone stimulation. Bone marrow had myeloid hyperplasia and reduced mature neutrophils. Recovery occurred in all 81 patients who passed the age of 5 years, except for one patient who is recovering at 6 1/2 years. The median age at recovery was 30 months; 95% recovered before 4 years. The estimated median duration of disease was 20 months. Neutrophil antibodies were detected early in the neutropenic phase by a combination of immunofluorescence and agglutination tests. Ten percent of these antibodies had specificity for NA1 or NA2. Ten of the 12 serum samples with a strong reaction in the flow cytometer reacted only with neutrophils. Two also reacted with an unidentified subpopulation (30%) of lymphocytes. Lymphocyte subsets were normal in 10 patients investigated, and abnormal levels of circulating immune complexes were detected in sera from 11 of 25 (44%) patients tested. Temporary remission was induced in all of eight patients who received intravenous IgG therapy. Autoimmune neutropenia of infancy is probably the most common cause of chronic neutropenia in infancy and early childhood, can be diagnosed by immunologic techniques, and requires only conservative management; spontaneous cure appears to be the rule.

Carbohydrate-specific antibodies in normal human sera. II. Structural definition of antigenic determinants.

IgG and IgM antibodies against several sugars have been characterized in sera of normal donors by passive hemagglutination and a quantitative hemagglutination inhibition test. These antibodies distinguish between the equatorial and axial OH groups at C2, C3, or C4 positions of the glycopyranose configuration, differences between the anomers, linkage types, changes in the primary alcohol group at C6, and OH substitution. In the examples of antibodies to mannose, galactose, and glucose investigated, specificities were usually directed against the beta-anomers. In disaccharides, the antibodies appeared to react only with 1 of the 2 sugar subunits, but unlike monosaccharides, the glycosidic linkages also seemed to be a part of the reaction site. Thus, the reacting moiety in gentiobiose was beta-D-glucopyranosyl with 1----6 linkage, in cellobiose with beta-D-glucopyranosyl with 1----4 linkage, in meliboise was alpha-D-galactopyranosyl with 1----6 linkage, and in lactose the reaction was directed against beta-D-galactopyranosyl with 1----4 linkage. In the maltose-dependent hemagglutination, alpha-D-glucose appeared to be the main reaction site. ManNAc exemplified the specificity determined by OH group substitution. Antibody to D-Fucose represented example of specificity evolving from substitution of the primary alcohol.

Carbohydrate-specific antibodies in normal human sera. I. Characterization of specificity for beta-D-glucose.

A glucose-dependent hemagglutinin, present in the serum of an apparently healthy donor, is shown to be a polyclonal IgM immunoglobulin with specificity for beta-D-glucopyranose configuration. The antibody did not agglutinate erythrocytes coated with hexoses differing from glucose at C2, C3, or C4 positions or lacking the primary alcohol group at C6 position. The hemagglutination reaction, quantitated in a continuous flow system, was inhibited by the addition of 6-deoxyglucose, 5-thioglucose, 2-deoxyglucose, 2-aminoglucose or 3-0 methylglucose. D-glucose, in beta- but not in alpha-configuration, attached by a glycosidic bond to another glucose or to an aglycone, was also inhibitory. A cross-reactivity was demonstrated between glucose and 6-deoxyglucose by antibody absorption and elution techniques. The donor's serum contained independent antibodies that reacted with erythrocytes coated with melibiose, gentiobiose, cellobiose, or N-acetylmannosamine. Further investigation revealed that antibodies are present in sera of all normal adults against erythrocytes coated with melibiose and N-acetylmannosamine and with high frequency against erythrocytes coated with gentiobiose, L-rhamnose, cellobiose, D-mannose, lactose, or D-galactose.