The major rheumatoid factor cross-reactive idiotype in rheumatic disease.

The major rheumatoid factor cross-reactive idiotype (RCRI), a tertiary structure formed by both light and heavy chains, is found on 60% of all monoclonal IgM kappa RFs. To determine if the RCRI is expressed in patients with rheumatic disease, we used polyclonal rabbit anti-idiotypic antibodies to detect RCRI in sera and in pokeweed mitogen cultures of blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). We detected increased expression of RCRI+, plasma cells in PWM cultures, and in sera from these patients. We have determined that some 7S IgM molecules from RF+RA patients are RCRI+, and can bind IgG in a sensitive RF ELISA. We have also observed that the CD5+ B cell subset, which is responsible for autoantibody production, generates RCRI+ antibodies. We review these data and discuss the relationship of the idiotypic network of interacting antibodies with rheumatic disease.

Development of the ability to make IgG and IgA in newborns and infants.

An understanding of the ontogeny of the human immune system may provide clues for methods to stimulate the immune response of neonates and young infants at an earlier age than is now possible. Knowledge of normal changes in immune response over time is necessary to evaluate infants for immunodeficiency.

Optimizing the approach to anemia in the preterm infant: is there a role for erythropoietin therapy?

OBJECTIVE: To define a subset of very low birth weight (VLBW) infants who might benefit from recombinant human erythropoietin (r-HuEPO) treatment. STUDY DESIGN: We reviewed the records for all VLBW (birth weight (BW) < or = 1500 gm) infants who were admitted to our nursery within the first 3 days of life between January 1991 and December 1994 and discharged alive. RESULTS: These infants received an average of 2.02 transfusions, far fewer than the 7 to 11 previously reported for VLBW infants. Infants with a BW of 1251 to 1500 gm received very few transfusions. More than three quarters of transfused infants received a transfusion in the first 2 weeks of life before r-HuEPO would be expected to be effective. Assigning units to individual infants and holding the units for 14 days, a practice adopted in our blood bank in 1993, resulted in a 44% decrease in donor exposures in infants receiving more than one transfusion. Holding assigned units for 30 days, a practice our blood bank has now adopted, should result in 56% of all transfused infants having a single donor exposure and 89% having one or two donor exposures. Cost-benefit analysis only supports routine use of r-HuEPO in infants weighing less than 750 gm. CONCLUSION: VLBW infants receive far fewer transfusions than the number previously reported. Assigning units to individual patients and holding those units for 30 days, together with efforts to minimize the need for transfusions make routine use of r-HuEPO unnecessary.

Pulse oximetry saturations in the first 6 hours of life in normal term infants.

The pulse oximetry saturation values and the average percentage of time that normal newborns spend at different saturation ranges in the first 6 hours of life were determined in a cross-sectional study. Pulse oximetry saturation values were measured for a single 20-minute period in 101 normal term newborns between 20 minutes and 6 hours of age. The 25th percentile saturation values in the first postnatal hour (range 91%-100%) were lower than those from the second postnatal hour (range 96%-100%) onward. There was no significant difference between the 50th percentile (range 96%-100%) and the 75th percentile (range 97%-100%) saturation values in all postnatal hours. The babies spent a majority of time with saturations > or = 96% in all postnatal hours. A newborn more than 20 minutes old who does not achieve a pulse oximetry saturation value of 96% over several minutes of observation may need evaluation or continuous monitoring.

Enzymatic activities in cultured human lymphocytes that dephosphorylate dolichyl pyrophosphate and dolichyl phosphate.

Enzymatic activities which dephosphorylate dolichyl phosphate (Dol-P) and dolichyl pyrophosphate (Dol-P-P) have been observed in membranes from cultured human lymphocytes. Neither activity requires divalent metals. Dol-P phosphatase is inhibited by inorganic phosphate but not by other phosphate-containing compounds. Dol-P-P phosphatase is inhibited by bacitracin but not by phosphate-containing compounds including the methylene analogue of pyrophosphate. These reactions are similar to those previously found in the cycle of bacterial wall peptidoglycan biosynthesis. A chemical synthesis of [32P]Dol-P and [32P]Dol-P-P is reported.

Expression of large quantities of rheumatoid factor major cross-reactive idiotype in the serum of adults with seropositive rheumatoid arthritis.

We quantified rheumatoid factor cross-reactive idiotype (RF-CRI) in whole serum from RF+ rheumatoid arthritis (RA) patients, using an inhibition enzyme-linked immunosorbent assay which is not affected by the presence of IgG. Serum from 16 RF+ RA patients contained 2-252 micrograms/ml RF-CRI (geometric mean */divided by SD 20.8 */divided by 5.2), while serum from 11 normal adults contained 1-16 micrograms/ml RF-CRI (geometric mean */divided by SD 3.9 */divided by 2.3). Serum from 8 of the RF+ RA patients contained RF-CRI at concentrations more than 2 standard deviations above the geometric mean in the normal subjects (greater than 21 micrograms/ml). Our results indicate that some RF+ RA patients express high concentrations of RF-RCRI and immunoglobulin molecules that express the RF-CRI may not be RF.

Effect of interferon-gamma and tumor necrosis factor on the expression of class I and class II major histocompatibility molecules by cultured human umbilical vein endothelial cells.

The expression of class I and class II major histocompatibility complex (MHC) molecules on the surface of cultured human umbilical vein endothelial cells (HUVEC) incubated with alpha tumor necrosis factor (TNF) or interferon-gamma (IFN-gamma) was determined by fluorescence flow cytometry. HUVEC were stained with fluorescein conjugated monoclonal antibodies (MoAbs) directed against monomorphic determinants of class I MHC molecules, HLA-A, B alpha chain (HLA-alpha) and beta-2-microglobulin (B2m), and class II MHC molecules, HLA-DR and HLA-DQ. HUVEC exposed to TNF for 96 hr increase their expression of class I MHC molecules two- to fourfold. Similarly, IFN-gamma increases the expression of class I MHC molecules two- to fivefold. No induction of HLA-DR or HLA-DQ was seen following exposure to TNF. IFN-gamma induces the appearance and markedly increases the expression of HLA-DR following 96 hr incubation. HLA-DQ was also induced by IFN-gamma but to a much lesser extent. TNF in combination with IFN-gamma enhances HUVEC expression of HLA-alpha, B2m, and HLA-DQ greater than that observed with either mediator alone. TNF and IFN-gamma reduced HUVEC expression of HLA-DR below the level observed with IFN-gamma alone. Indomethacin (INDO) does not effect expression of HUVEC class I or class II MHC molecules induced by TNF, IFN-gamma, or the combination of these mediators. These immune mediators produce unique and common effects on HUVEC, and therefore may act by at least two separate mechanisms, independent of the cyclooxygenase pathway, which regulate HUVEC expression of class I and class II MHC surface molecules.

Leishmania amastigotes: resistance to complement-mediated lysis is not due to a failure to fix C3.

Amastigote forms of Leishmania major are sensitive to lysis by fresh serum, whereas those of L. donovani are resistant. To understand the basis for this resistance we have examined the interaction of complement with amastigotes of seven strains of leishmania. Complement activation was determined by measuring the ability of amastigotes to consume complement from normal serum and by identifying parasite surface-bound C3. All of the strains that were tested activated complement, including both those that are resistant and those that are susceptible to inactivation by fresh serum. Complement consumption by amastigotes was measured as a decrease in the ACH 50 titers of serum exposed to parasites. L. major, L. donovani, and L. mexicana mexicana (strain 1VLM) amastigotes decrease titers by 35.7, 33.5, and 40.3%, respectively. The binding of C3 to amastigotes was judged qualitatively by immunofluorescence and quantitatively by a C3 radiobinding assay. L. major amastigotes bind an average of 6.6 X 10(4) molecules of C3 per parasite. L. mexicana amazonensis, L. mexicana mexicana, and L. donovani bind an average of 3.9 X 10(4), 5.9 X 10(4), and 3.7 X 10(4) molecules, respectively. In all cases, C3 binding is the result of alternative pathway activation requiring Mg++ but not Ca++. Amastigotes of the disseminating strains of leishmania represent the first example of a group of protozoa that activate early complement components leading to fixation of C3, but that are resistant to inactivation by complement.

Expression of the major rheumatoid factor cross-reactive idiotype in juvenile rheumatoid arthritis.

The major rheumatoid factor cross-reactive idiotype (RF-CRI), defined by prototypic monoclonal IgM rheumatoid factors, is expressed in high frequency by pokeweed mitogen-derived plasma cells from patients with rheumatoid arthritis who express RF in their sera. Unlike adults with rheumatoid arthritis, most patients with juvenile rheumatoid arthritis are seronegative for RF, as detected by classic IgG binding assays. We report that approximately 50% of seronegative patients with juvenile rheumatoid arthritis express the RF-CRI in high frequency among their pokeweed mitogen-derived plasma cells, and that approximately 33% of patients express the RF-CRI in high titer in their sera. The possible mechanisms for expression of an idiotypic marker of RF without expression of IgG binding activity by classic assays are discussed.

Expression of the major rheumatoid factor cross-reactive idiotype in pediatric patients with systemic lupus erythematosus.

Rheumatoid factor cross-reactive idiotype (RF-CRI) is expressed in high concentrations in the sera of some patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). To determine if RF-CRI is specifically expressed in rheumatic disease or if it is secondary to polyclonal B-cell activation, we examined sera of 23 children with SLE, 16 adolescents with infectious mononucleosis (IM), and age-matched pediatric controls for RF-CRI expression. Concentrations of RF-CRI in serum, determined by an inhibition ELISA, were 24 +/- 17 micrograms/ml (mean +/- SD) in 25 normal children, 31 +/- 17 in 16 young adults with IM, and were significantly increased, 70 +/- 80 micrograms/ml, in the 23 children with SLE (p less than 0.036). Eleven of 23 SLE patients had serum RF-CRI greater than the mean +/- 2 SD for normal children. Ten of 23 SLE sera contained IgM rheumatoid factor (RF) activity. One patient with IM had a borderline elevated RF-CRI level, and 5 IM patients had RF in their sera. The serum IgM concentrations in sera were: SLE (192 +/- 93 mg/dl) and IM (234 +/- 77 mg/dl) sera. These levels were significantly elevated compared to controls (132 +/- 44 mg/dl), p less than 0.031 for SLE and p less than 0.001 for IM, suggesting that polyclonal activation of B cells was present in SLE and IM patient groups. Increased expression of RF-CRI in the SLE patients correlated directly with high titer anti-DNA antibody values (r = 0.3965, p less than 0.05) and RF activity when human IgG (r = 0.5026, p less than 0.05) was used as the RF binding substrate and inversely with serum C3 levels (r = 0.3925, p less than 0.05). RF-CRI expression did not correlate with RF that bound rabbit (r = 0.3123, p greater than 0.05). Increased serum RF-CRI expression is not a result of polyclonal B-cell activation. RF-CRI may be selectively up-regulated in patients with SLE.

The rheumatoid factor cross-reactive idiotype in juvenile rheumatoid arthritis: role of the CD5-positive B cell.

The major rheumatoid factor cross-reactive idiotype (RCRI), which was defined by prototypic monoclonal IgM rheumatoid factors (RF) from Waldenstrom's macroglobulinemia patient Wa, is present on 60% of all monoclonal IgM RF paraproteins. The RCRI is expressed in high frequency by pokeweed mitogen-derived plasma cells (PWM-PCs) and in high concentration in the sera from adults with rheumatoid arthritis (RA) who express RF in their sera. Unlike adults with RA, most children with juvenile rheumatoid arthritis (JRA) are seronegative for RF as detected by classic IgG binding assays. In the experiments summarized herein, we demonstrated that approximately 2/3 of JRA patients who are seronegative for RF, express the RCRI in high concentration in their sera and in high frequency among their PWM-PCs. Expression of this idiotype could not be attributed to expression of hidden IgM RF, or IgA RF, and may be expressed on a parallel set of immunoglobulin molecules, related to RFs, but lacking the ability to bind to IgG. There is an increased number of circulating CD5+ B cells in patients with JRA but there was no significant relationship between CD5+ B cell numbers and serum RCRI concentration, suggesting that in this disease, RCRI bearing immunoglobulins may also be produced by non-CD5+ B cells or by a small subset of CD5+ B cells.

Enzymatic utilization of P1-di-N-acetylchitobiosyl P2-dolichyl pyrophosphate and its chemical synthesis.

Fully acetylated chitobiose was treated with phosphoric acid to give a mixture of products from which 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta- D-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-alpha,beta- D-glucopyranosyl phosphate (Ac(3)GlcNAc-Ac(2)GlcNAc-P) was isolated by preparative thin layer chromatography. Treatment of this compound with P(1)-diphenyl P(2)-dolichyl pyrophosphate gave an acetylated pyrophosphate diester, which was purified chromatographically and deacetylated. The product, P(1)-di-N-acetylchitobiosyl P(2)-dolichyl pyrophosphate (Dol-P-P-GlcNAc-GlcNAc), was readily separated from P(1)-2-acetamido-2-deoxy-alpha-D-glucopyranosyl P(2)-dolichyl pyrophosphate (Dol-P-P-GlcNAc) by thin layer chromatography in several solvent systems. Addition of this compound to human lymphocyte membrane preparations led to the formation of a mannose-containing derivative which appears to be an oligosaccharide phospholipid, as judged by its behavior on DEAE-cellulose chromatography and by its hydrolysis to give an oligosaccharide containing more than four monosaccharide units.

Antibody-mediated enhancement of respiratory syncytial virus infection in two monocyte/macrophage cell lines.

Monoclonal antibodies (MAbs) specific for two surface glycoproteins of respiratory syncytial virus (RSV) were found to enhance RSV infection in two macrophagelike cell lines (P388D1 and THP-1). MAbs to an irrelevant antigen (pneumococcal polysaccharide) and to the nucleocapsid of RSV did not enhance infection. Blocking either the Fc segment of the monoclonal antibody of the Fc receptor on the cells diminished the enhancement, suggesting that this phenomenon involves attachment of the monoclonal antibody to the virus followed by attachment of the Fc of this complex to the Fc receptor on the cells. These data indicate that antibody-mediated enhancement of RSV infection can occur in vitro in macrophages. This enhancement may contribute to the pathogenesis of RSV bronchiolitis and the more severe RSV disease seen in recipients of formalin-inactivated RSV vaccine.

Hidden rheumatoid factor and Wa idiotype expression in juvenile rheumatoid arthritis.

The major rheumatoid factor cross-reactive idiotype, which was defined by prototypic monoclonal IgM rheumatoid factors from Waldenstrom's macroglobulinaemia patient Wa, is present on 60% of all monoclonal IgM RF paraproteins. One-third of patients with juvenile rheumatoid arthritis (JRA), who are seronegative for classic IgM rheumatoid factor (RF), express the Wa idiotype in high titre in their sera. To determine if the Wa idiotype is present on hidden rheumatoid factors in JRA patient sera, we studied hidden RF expression by both ELISA and haemolytic assay techniques. The majority of JRA sera with increased concentrations of the Wa idiotype did not have increased RF activity nor hidden RF activity. In some JRA patients, the Wa idiotype may be expressed on a parallel set of immunoglobulin molecules, related to RFs, but lacking the ability to bind to IgG.

Umbilical cord blood lacks circulating B lymphocytes expressing surface IgG or IgA.

In vitro umbilical cord blood B lymphocytes fail to form IgG and IgA secreting plasma cells when stimulated with Pokeweed mitogen. Since previous investigators have found percentages of B lymphocytes expressing surface IgG or surface IgA comparable to those seen in adults, this implies a defect in umbilical cord blood B-lymphocyte function. We have examined surface Ig expression on umbilical cord blood B lymphocytes by flow cytometry under conditions in which serum derived Ig are rigorously excluded. Under these conditions no B lymphocytes expressing surface IgG or IgA, which should serve as precursors for IgG and IgA secreting plasma cells, were observed. This finding was confirmed by comparing the ratio of mRNA levels for immunoglobulin gamma-chain to mu-chain in mononuclear cells by quantitative mRNA-based PCR. The ratio in umbilical cord mononuclear cells was 10-fold less than that seen in adult cells. The inability of newborn peripheral blood to form IgG and IgA plasma cells may result from an absence of appropriate precursor cells and not a defect in B lymphocyte maturation.

Detection of the major rheumatoid factor cross-reactive idiotype precedes human IgG binding activity in a patient with polyarticular juvenile rheumatoid arthritis.

A patient with polyarthritis and subcutaneous nodules was studied for expression of rheumatoid factors (RF) by numerous techniques for 5 months after presentation. Assays for RF binding human or rabbit IgG in whole sera, assays for hidden RF binding human or rabbit IgG, and assays for expression of the major RF cross-reactive idiotype (RCRI) in whole sera or by pokeweed mitogen induced plasma cells were performed (PWM-PC). In this patient, increased expression of RCRI in sera and among PWM-PC preceded detectable RF binding human IgG, and paralleled hidden RF and RF binding rabbit IgG expression.

Seronegative rheumatoid arthritis, rheumatoid factor cross reactive idiotype expression, and hidden rheumatoid factors.

The major rheumatoid factor cross reactive idiotype (RCRI), defined by prototypic monoclonal rheumatoid factors (RFs), is expressed as a dominant idiotype by pokeweed mitogen induced plasma cells obtained from seropositive (RF+) patients with rheumatoid arthritis (RA). Some patients who meet clinical diagnostic criteria for RA set by the American Rheumatism Association fail to express RFs at any time during their clinical course. To determine if seronegative (RF-) patients with RA, so designated by the latex fixation, Rose-Waaler classic binding assays, or a RF enzyme linked immunosorbent assay (ELISA), express the RCRI in the absence of detectable RFs we examined pokeweed mitogen plasma cells from these patients by indirect immunofluorescence. In addition, we used an inhibition ELISA to detect RCRI bearing molecules in the sera of RF- patients with RA. Five of 10 RF- patients with RA had a high prevalence of RCRI+ plasma cells (16-49% of total pokeweed mitogen plasma cells in culture). Six of 20 RF- patients with RA had high serum concentrations of molecules marked by the RCRI, equivalent to 21-110 micrograms/ml of RCRI+ reference monoclonal IgM RF. Four of five patients who expressed the RCRI in high prevalence in pokeweed mitogen plasma cells, also demonstrated high concentrations of RCRI in their sera detected by inhibition ELISA. There was significant concordance of RCRI expression determined by the two different assays. Four RF- patients with RA who expressed RCRI in their whole sera had hidden RFs detected in their 19S and, in one case, 7S serum fraction. Detection of RF related molecules in whole sera by the expression of RCRI in RF- patients with RA identifies a subgroup of RF- patients with RA who possess hidden RFs. Some RF- patients with RA can express the major RCRI in pokeweed mitogen plasma cells and in their sera and therefore are related to patients with prototypic Waldenstrom's macroglobulinaemia, who produce RCRI+ 19S IgM monoclonal RFs.

Leishmania species: mechanisms of complement activation by five strains of promastigotes.

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.