Platelet cold agglutinins: a flow cytometric analysis.

Spontaneous EDTA-independent cold platelet agglutination is a rare phenomenon that produces pseudothrombocytopenia when blood samples are analyzed in automated cell counters. We report a case of platelet cold agglutinins and an analysis by flow cytometry. A 49 year old woman presented with abnormal vaginal bleed secondary to uterine fibroids. Platelet clumping was observed in blood samples taken in EDTA-, heparin- and citrate-containing tubes. In flow cytometric tests, patient serum agglutinated 16% of normal platelets at 22 degrees C, and 7% of platelets after incubation at 37 degrees C; in contrast, 3% and < 1% of platelets were agglutinated at 22 and 37 degrees C, respectively, after incubation with normal serum. Minimal agglutination (< 10%) was observed with patient serum at a titre of 1:5 or at temperatures > 30 degrees C. After incubation at 4 degrees C, IgM antibody and C3 were increased on the patient's platelets; no significant amount of IgM or C3 was detected on normal platelets. The specificity of the platelet cold agglutinin was determined by competitive inhibition by monoclonal anti-CD41(GPIIbIIIa). Before the addition of monoclonal antibody, patient's serum agglutinated 16% of normal platelets at 22 degrees C; after addition of anti-CD41 only 2% of the platelets were agglutinated. This blocking effect was not observed with anti-CD42. The patient's platelets functioned normally as determined by CD62 and CD63 expression in response to thrombin, normal platelet aggregation in response to collagen, ADP, and ristocetin, and a normal template bleeding time. In summary, platelet agglutination by a platelet cold agglutinin was quantitated by flow cytometry, the responsible antibody was characterized as a low titre IgM with minimal activity > 30 degrees C, and competitive binding studies supported the GPIIbIIIa complex as the binding site for the antibody. Since the antibody did not affect platelet function, we believe that these patients will not suffer complications from their platelet cold agglutinin, but it could pose a problem under circumstances such as cardiac surgery with hypothermia.     

The immunosuppressive effect of monoclonal anti-Lyt-1.1 antibodies in vivo

Monoclonal anti-Lyt-1.1 alloantibody was produced as tissue culture supernatant and administered to mice. The antibody, given intraperitoneally, resulted in the suppression of all T cell functions studied, but was without direct effect on B cells. Thus, skin and tumour allograft survival was prolonged and there was suppression of the delayed-type hypersensitivity response; T cell help inthe anti-sheep red blood cell antibody response, responder cells in the mixed lymphocyte reaction (MLR), leucoagglutinin-responsive cells, cytotoxic T cell (Tc) function and the induction of Tc were either totally or partially suppressed, all these responses being mediated by Lyt-1+2- or Lyt-1+2+ cells in CBA/H mice. By contrast, there was no inhibitory effect on the MLR-stimulating or lipopolysaccharide-responsive cells. The administration of the anti-Lyt-1.1 antibody was accompanied by a depletion of Lyt-1.1+ T cells from both spleen and lymph node. These studies indicate that the monoclonal anti-Lyt-1.1 antibody is active in vivo with a selective effect on T cells. The results also have important implications for studies of T cell interactions in the mouse in vivo, and for similar studies in man.     

Creating a mission-based reporting system at an academic health center.

The authors developed a Web-based mission-based reporting (MBR) system for their university's (UC Davis's) health system to report faculty members' activities in research and creative work, clinical service, education, and community/university service. They developed the system over several years (1998-2001) in response to a perceived need to better define faculty members' productivity for faculty development, financial management, and program assessment. The goal was to create a measurement tool that could be used by department chairs to counsel faculty on their performances. The MBR system provides measures of effort for each of the university's four missions. Departments or the school can use the output to better define expenditures and allocations of resources. The system provides both a quantitative metric of times spent on various activities within each mission, and a qualitative metric for the effort expended. The authors report the process of developing the MBR system and making it applicable for both clinical and basic science departments, and the mixed success experienced in its implementation. The system appears to depict the activities of most faculty fairly accurately, and chairs of test departments have been generally enthusiastic. However, resistance to general implementation remains, chiefly due to concerns about reliability, validity, and time required for completing the report. The authors conclude that MBR can be useful but will require some streamlining and the elimination of other redundant reporting instruments. A well-defined purpose is required to motivate its use.     

Chimeric Fc receptors identify immunoglobulin-binding regions in human FcγRII and FcϵRI

FcγRII and Fc?RI are functionally distinct cell surface receptors for immunoglobulin (Ig); FcγRII binds IgG with low affinity, whereas Fc?RI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig-like domains with 38% amino acid identity. Chimeric receptors derived from human FcγRII and FcγRI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in FcγRII and IgE in Fc?RI. Firstly, a chimeric form of the Fc?RI α chain was produced by replacing the transmembrane region and cytoplasmic tail with that of FcγRII. This mutant α chain could be expressed on the cell surface independently of associated β and γ subunits, and retained high-affinity IgE binding, indicating that the extracellular region of the FcγRI α chain is sufficient for high-affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both FcγRII and FcγRI, chimeric receptors were generated by exchanging the first extracellular domains between FcγRII and the α chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of FcγRII and FcγRI; these identified a single region of IgG binding in FcγRII located between residues Ser¹³⁶ to Val¹⁶⁹, and at least three independent IgE binding regions in the FcγRI α chain, between residues Trp⁸⁷ to Lys¹²⁸, Tyr¹²⁹ to Asp¹⁴⁵, and Ser¹⁴⁶ to Val¹⁶⁹.     

Inhibition of hyperacute transplant rejection by soluble proteins with the functional domains of CD46 and FcgammaRII

BACKGROUND: Recombinant soluble forms of complement regulatory molecules, including the human complement regulatory protein CD46 (rsCD46), have been shown to inhibit hyperacute transplant rejection (HAR) and protect against complement-mediated inflammatory tissue damage. Similarly, recombinant soluble forms of the immunoglobulin receptor FcgammaRII (rsFcgammaRII) can attenuate antibody-mediated inflammatory responses. We have produced and tested the function of novel recombinant chimeric proteins that incorporate the functional domains of both CD46 (membrane cofactor protein, MCP) and the low affinity human IgG receptor FcgammaRII (CD32). METHODS: Two recombinant soluble chimeric proteins (CD46:FcR and FcR:CD46) were designed and produced using a human cell expression system. Their ability to protect cells against complement-mediated lysis (through the CD46 domain) and bind human IgG (through the Fc receptor domain) was assessed in vitro. They were also tested in vivo in the rat reverse passive Arthus reaction and a murine model of hyperacute cardiac transplant rejection. RESULTS: In vitro, the functional domains of the chimeric proteins each retained their activity. In vivo, the serum half-life of the recombinant chimeric proteins in mice was more than either rsCD46 or rsFcgammaRII. In the rat reverse passive Arthus reaction, intradermal injection of each recombinant protein substantially reduced inflammatory skin edema (>50%) and polymorphonuclear neutrophil infiltration (>90%). In the hyperacute rejection model, i.v. treatment with FcR:CD46 prevented complement-mediated rejection, macroscopic bruising, edema, and thrombosis more effectively than rsCD46. CONCLUSIONS: CD46/FcgammaRII bifunctional proteins have an improved ability to control complement-mediated hyperacute graft rejection and have therapeutic potential in other conditions involving antibody-mediated inflammation.     

Reiter's syndrome following intravesical BCG immunotherapy

A 71 year old woman developed conjunctivitis, asymmetrical oligoarthritis, and cystitis (Reiter's syndrome) secondary to intravesical BCG treatment for transitional cell carcinoma of the bladder. She received oral prednisolone, izoniazid, and pyridoxine and made a full recovery. Increasing use of BCG as immunotherapy will lead to an increase in the incidence of BCG associated reactive arthritis. Prompt recognition and early diagnosis will facilitate treatment and recovery.     

Prognostic performance of proteomic testing in advanced non-small cell lung cancer: a systematic literature review and meta-analysis

Abstract

Objective

Timely assessment of patient-specific prognosis is critical to oncology care involving a shared decision-making approach, but clinical prognostic factors traditionally used in NSCLC have limitations. We examine a proteomic test to address these limitations.     

Development of a Mycobacterium bovis intranasal challenge model in mice

Tuberculosis caused by infection with Mycobacterium tuberculosis or Mycobacterium bovis remains one of the most important infectious diseases of man and animals. The current vaccine, M. bovis bacille Calmette-Guérin (BCG) demonstrates variable efficacy in humans and cattle, and so an urgent need exists for a new vaccine to replace or supplement BCG. Novel vaccine development requires the availability of a suitable animal model in which to test potential vaccine candidates. Models for tuberculosis vaccine development include mice, guinea pigs, cattle and non-human primates. Murine models provide an economical and easily manipulated tool, but the natural aerosol infection route requires extensive facilities, equipment and validation. We sought to develop a logistically simpler intranasal M. bovis infection model for use in vaccine development for bovine tuberculosis. Intranasal M. bovis infection model in mice demonstrated distinct airway associated, dose related pathology, and was strikingly more virulent than previously employed intravenous infection with M. bovis. BCG vaccination of intranasal challenged mice induced 2 logs of protection with similar kinetics as those displayed in M. tuberculosis aerosol infection models. In conclusion, we report the development of a virulent, robust, stringent, physiological and inexpensive M. bovis intranasal infection model for the screening of potential vaccine candidates against bovine tuberculosis.