Soluble circulating Fc gamma receptors in human serum. A new ELISA assay for specific and quantitative detection.

We have developed a simple, rapid, and sensitive enzyme-linked immunoadsorbent assay (ELISA) to measure soluble cell-free human Fc gamma receptor (Fc gamma R) in serum. This assay is based on the use of two monoclonal antibodies directed against different epitopes expressed on the same low avidity human Fc gamma R (CD16), which is present on polymorphonuclear leukocytes, macrophages and NK cells. This sandwich ELISA, which can measure 2 nM concentration of Fc gamma R, has enabled us to demonstrate the presence and to measure the level of soluble cell-free human Fc gamma R (CfH-Fc gamma R) in normal human serum.

Posttransplantation plasmacytic proliferations related to Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), which was originally detected in Kaposi's sarcoma, also has been found in primary effusion lymphomas (PELs) and some cases of multicentric Castleman's disease. We describe two transplant recipients who developed Kaposi's sarcoma and a spectrum of non-neoplastic lymphoproliferative disorders that show pronounced plasmacytic and plasmacytoid features. The first patient had recurrent pleural effusions and Castleman's disease-like changes in lymph nodes. The second patient had systemic lymphadenopathy and hepatosplenomegaly secondary to diffuse infiltration by polyclonal plasma cells and plasmacytoid B lymphocytes that clinically mimicked Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease. In both cases, KSHV DNA was detected by polymerase chain reaction and Southern blotting, and KSHV vIL-6 protein expression was identified in affected tissues by immunohistochemical localization. In contrast, no evidence of KSHV coinfection was detected in any of 31 EBV-related posttransplant lymphoproliferative disorders or 112 non-PEL lymphomas tested. The pathologic findings in these two patients were not representative of malignancy by morphologic, immunophenotypic, or molecular criteria. This study underscores the marked propensity for hematolymphoid proliferations associated with KSHV infections to show plasmacytic features. Additionally, this study describes use of an antibody reactive against KSHV vIL-6 that can readily detect a subpopulation of KSHV-infected hematopoietic cells.

Persistent hypersplenism early after liver transplant: the role of splenectomy.

BACKGROUND: Transient thrombocytopenia is common after liver transplantation, but persisting thrombocytopenia worsens the prognosis after transplant. METHODS: Two patients underwent splenectomy for persistent thrombocytopenia early after liver transplantation. The first patient had a platelet count of 17,000/mm3 on postoperative day (POD) 6; her hemoglobin and white blood cell counts were normal. Work-ups including bone marrow aspiration, Coombs test, and antiplatelet antibody test were negative. On POD 9, she had abdominal bleeding with a platelet count of 17,000/mm3 despite repeated platelet transfusions, and splenectomy was done. The second patient had a platelet count of 3000/mm3 on POD 14, white blood cell was 1600/mm3, and hemoglobin was 7.7 g/dl. Bone marrow biopsy revealed hypercellular marrow. Because his platelet count remained at 2000/mm3 despite empiric treatment with intravenous immune globulin and methylprednisolone, splenectomy was performed. RESULTS: The first patient's platelet count rose to 155,000/mm3 by POD 8. The second patient's platelet count reached 210,000/mm3 on POD 5. Neither patient has had an episode of thrombocytopenia at 36 and 32 months after splenectomy. CONCLUSIONS: Splenectomy can be used after liver transplantation for severe, persistent thrombocytopenic states that cannot be attributed to sepsis, intravascular coagulation, immunological causes, or drug effects.

Comparative analysis of HLA-B 8101 by serology, isoelectric focusing, and sequence-based typing.

We describe a bone marrow donor evaluation on a twenty-four-year-old patient from Honduras, Central America. The patient's phenotype included a HLA-B7v, with serologic reactivity to HLA- B7, 8101, and 48, and was consistent with, HLA-B*8101. One-dimensional isoelectric focusing (IEF) identified HLA-B 7.1, a low frequency isotype that has previously been associated with the HLA-B7 variant, B*0705. To further classify this allele, sequence based typing (SBT) was performed, confirming HLA-B*8101 sequence homology. The diagnostic evaluation of this case illustrates the correlation of sequenced based typing and isoelectric focusing in defining HLA Class I antigen characteristics and the use of IEF in screening for rare and novel alleles.

An immunological syndrome featuring transverse myelitis, Evans syndrome and pulmonary infiltrates after unrelated bone marrow transplant in a patient with severe aplastic anemia.

A patient with severe aplastic anemia underwent a matched unrelated bone marrow transplant, following which he developed a complex autoimmune syndrome. This featured transverse myelitis, immune mediated Coombs positive hemolytic anemia and immune thrombocytopenia (Evans syndrome), pulmonary infiltrates, eosinophilia, muscle pains and cramps and lichenoid dermatitis all of which may represent manifestations of graft-versus-host disease as they showed response to immunosuppression. Thus, although immune-mediated cytopenias after an allogeneic bone marrow transplant are rare, they should be considered as a possible cause of cytopenia in post-transplant patients.

Interphase FISH analysis of sex-mismatched BMT utilizing dual color XY probes.

Interphase FISH analysis, utilizing dual color XY probes, was performed on 27 patients following allogeneic sex-mismatched bone marrow transplantation and on 31 controls. Of the 123 167 examined interphase nuclei, 63 318 were from 19 of the 21 patients (54 specimens) who engrafted, 31 827 from five of the six patients (29 specimens) who relapsed (four) or failed to engraft (one) and 24 703 from the 31 control specimens. In patients who engrafted, the mean percentage of host cells was 0.26% between day 29 and 5 years following BMT. Microchimerism of 0.7% or less than 1-5 years following BMT was not predictive of relapse. Interphase FISH analysis predicted relapse or failure of engraftment in five of the six evaluable patients. In three of five patients both conventional cytogenetics and interphase FISH of bone marrow cells provided important information regarding engraftment status and degree of chimerism.

A pilot study of allogeneic bone marrow transplantation using related donors stimulated with G-CSF.

Growth factor administration to donors prior to bone marrow (BM) harvesting results in an enrichment of the graft for myeloid precursors. In animals, growth factor-primed BM has a higher repopulating ability than untreated BM. Ten patients received an HLA-identical sibling, allogeneic transplant using granulocyte colony-stimulating factor (G-CSF)-stimulated BM. Stimulation consisted of G-CSF at 10 microg/kg/day for 2 days prior to harvest. Patients were transplanted for various benign and malignant hematological conditions. The GVHD prophylaxis consisted of cyclosporine, methotrexate and/or prednisone. Compared to untreated historical control BM, stimulated BM infusions contained similar number of nucleated cells (mean +/- s.d.: 3.5 +/- 1.5 vs 4.0 +/- 0.9 x 10(8)/kg), CD34+ cells (mean +/- s.d.: 7.5 +/- 3.0 vs 9.4 +/- 6.7 x 10(6)/kg), and CD3+ cells (mean +/- s.d.: 129 +/- 30 vs 190 +/- 59 x 10(6)/kg) but higher numbers of granulocyte-macrophage colony-forming units (mean +/- s.d.: 20 +/- 12 vs 96 +/- 34 x 10(4)/kg). Patients receiving stimulated BM had prompt and stable engraftment of white cells and platelets. On average they attained an ANC of > or = 1 x 10(9)/l 9 days earlier and a platelet count of > or = 20 x 10(9)/l 6 days earlier than historical controls receiving unstimulated HLA-identical sibling BM. Hospitalization was shortened by a mean of 10 days and transfusion requirements were modest. None of the patients developed severe GVHD or disease relapse. Two patients died of severe VOD post-BMT and thus were unevaluable for platelet engraftment. A third patient died of TTP on day 76 post-BMT. Seven patients are alive and well 49-585 days post-BMT. Stimulated BM may provide a valuable alternative to allogeneic BM and PBSC transplants. Ideal stimulation regimens need to be investigated.

Predictive value of left ventricular ejection fraction in stem cell transplantation.

We evaluated predictive value of left ventricular ejection fraction at rest (REF) and its increment with exercise (deltaEF) on autologous and allogeneic stem cell transplantation mortality. In a 7 year period, a total of 163 patients evaluated for stem cell transplantation were studied. All were followed for at least 3 months after the transplant. REF was discriminatory for peritransplant mortality only in younger (<43 years) patients (n = 66), particularly those who underwent autologous transplantation (n = 30). Resting ejection fraction was not a discriminator for early death in any other subgroup. Cardiac reserve (deltaEF) was significantly lower in patients (n = 35), who died early. The finding was most prominent in younger patients who underwent autologous transplantation (n = 26). Combination of decreased REF and low deltaEF (n = 18) was associated with high peritransplant mortality (56%), after both autologous and allogeneic transplantation. A low REF with an appropriate deltaEF (n = 43) was associated with a 19% peritransplant mortality and no deaths in the autologous group. These observations indicate that resting ejection fraction is of only limited value for pretransplant evaluation. However, measurement of cardiac reserve during exercise can provide important prognostic information before stem cell transplantation.

Barriers to mental health service use among hematopoietic SCT survivors.

This study examined barriers to mental health service use and the demographic, medical and psychosocial correlates of these barriers among hematopoietic SCT (HSCT) survivors. A sample of 253 HSCT survivors who were 1 to 3 years posttransplant completed measures of demographic, physical, psychological and social characteristics as well as a newly modified measure of barriers to mental health service use. Only 50% of distressed HSCT survivors had received mental health services. An exploratory factor analysis of the barriers to mental health service use scale yielded four factors: scheduling barriers, knowledge barriers, emotional barriers and illness-related barriers. Patients with higher social constraints (perceived problems discussing the illness experience with significant others) reported higher levels of all four types of barriers. General distress and transplant-related posttraumatic stress symptoms were positively associated with emotional, knowledge and illness-related barriers to mental health service use, whereas physical and functional well-being were inversely associated with these barriers. Having more knowledge barriers and more emotional barriers predicted a lower likelihood of receiving mental health services, as did lower levels of education and general distress. Results suggest that a significant number of HSCT survivors may benefit from education about mental health services that is tailored to individual barriers.

Long-term follow-up after allogeneic granulocyte colony-stimulating factor--primed bone marrow transplantation.

Granulocyte colony-stimulating factor (G-CSF) priming increases the number of progenitor cells in harvested bone marrow (BM) and has been used for allogeneic transplantation. Primed bone marrow (pBM) seems to offer faster engraftment than steady-state BM, but the stability of such engraftment has been questioned. The incidence of graft-versus-host disease (GVHD) and disease relapse after pBM, compared with such incidence after BM or peripheral blood progenitor allotransplantation, has not been established. We studied the long-term outcome (median follow-up, 24 months) of sibling matched allografting with G-CSF pBM. Seventeen patients received pBM from matched sibling donors primed with G-CSF 10 microg/kg per day for 2 days before BM harvest. Conditioning consisted of total body irradiation and cyclophosphamide (CY); busulfan and CY; or total lymphoid irradiation, CY, and antithymocyte globulin. All infused grafts contained > or = 3.5 to 4 x 10(8) mononuclear cells per kilogram. Ten of 17 patients received methotrexate as part of their GVHD prophylaxis. International Bone Marrow Transplant Registry definitions for engraftment were used. Control subjects consisted of 112 consecutive patients who received allogeneic transplantation at our institution with steady-state BM; control subjects for length of hospitalization consisted of the subset of patients who underwent transplantation during 1996. Neutrophil engraftment occurred a median of 7 days earlier in primed bone marrow transplantation (pBMT) patients when compared with steady-state BMT patients; this shortened hospitalization by a median of 11 days. The peritransplant mortality rate was 18% in pBMT patients and 25% in BMT patients (not significant). The rate of GVHD of grade > II and the rate of relapse were almost identical in pBMT and BMT patients (GVHD: 18% and 19%, respectively; relapse: 14% and 13%, respectively). There were 4 transplant-related deaths within the first 100 days; 1 patient died of disease relapse on day 470. Twelve patients remained alive on days 430 through 1522 after BMT. Results showed that pBM allografts resulted in more rapid engraftment and shorter hospitalization. All patients maintained stable donor engraftment. In this cohort of patients, G-CSF pBMT resulted in rates of GVHD, disease relapse, and peritransplant mortality that were similar to those produced by conventional BMT.

Collection of autologous PBSC in patients with polycythemia vera.

BACKGROUND: Allogeneic stem-cell transplantation (SCT) can eradicate myelofibrosis (MF), but is limited by donor availability and toxicity. We previously reported normalization of counts and resolution of MF after ablative, syngeneic SCT in spent phase polycythemia vera (PV). Hence, GvL is not required to eradicate MF. Autologous SCT may advance treatment for spent phase PV by restoring effective hematopoiesis. The influence of organomegaly, myelosuppression and MF on PBSC collection has not been studied in the setting of PV. METHODS: Sixteen patients with PV underwent PBSC collection. Mobilization was with filgrastim alone, with a target cell content of 2.5 x 10(6) CD34(+)/kg. All myelosuppression was discontinued 2 weeks prior to collection. RESULTS: Median ages at diagnosis and collection were 47 and 57 years, respectively. Organomegaly, MF and use of myelosuppressive therapies were present in 10 (63%), 4 (25%) and 7 (44%) patients. Median total nucleated cells (TNC) and CD34(+) counts were 8.3 x 10(8)/kg and 4.98 x 10(8)/kg. MF had an adverse effect on TNC (p=0.05) but not on the CD34(+) content. Time from diagnosis and the use of myelosuppresion had no influence on TNC and CD34(+) contents. Four patients had CD34(+) contents <2.5 x 10(6)/kg. Complete blood count (CBC) parameters were not predictive of CD34(+) content. DISCUSSION: Autologous PBSC collection is feasible in PV several years after diagnosis. Organomegaly and MF are not absolute contraindications for collection. Discontinuing myelosuppresion for 2 weeks before mobilization appears sufficient to collect adequate numbers of CD34(+) progenitors.

Human leukocyte antigens (HLA)-Cw as prognostic indicators in autologous transplantation for lymphoma.

The human leukocyte antigens (HLA) function as transplantation antigens and as markers in disease association. Disparity at the HLA A, B, Cw, and DR loci in allogeneic stem cell transplants results in an increased incidence of graft-versus-host disease, graft rejection, and decreased survival. HLA class I loci A, B, and Cw also function as ligands for natural killer (NK) cell receptors in an interaction that predominantly inhibits cytolysis of target antigens. This HLA-NK cell inhibitory function is required for protection against auto-aggression, and is of unclear significance in other clinical settings. Furthermore, the prevention of auto-aggression is HLA molecule specific as demonstrated by the association of specific HLA types with autoimmune diseases. It is not known whether the HLA molecules might serve as markers for outcome in autologous transplants. We investigated an association of HLA class I molecules and early transplant outcome in a cohort of patients who underwent autologous transplantation for the treatment of lymphoma. In this retrospective study, HLA class I molecules were analyzed to determine whether they affect transplant outcome. HLA typing was performed by microlymphocytotoxicity assays. Factors such as age, sex, disease type, lactate dehydrogenase (LDH), cell dose, type of graft, and transfusion events were reviewed. Outcome was defined as death (or survival) at 6 months from the date of transplant. HLA-Cw8 was significantly associated with poor outcome (odds ratio = 18 and 9.3, p = 0.01 and 0.02 in homozygous and all patients, respectively). The HLA-A and B locus molecules were not associated with outcome. Age, sex, elevated LDH, and cell dose were not associated with outcome. A blood progenitor cell dose of greater than 6 x 10(8) nucleated cells/kg was favorably associated with outcome (p = 0.08). The number of transfusions received was not associated with outcome. In the multivariate analysis of HLAs and factors associated with outcome, HLA-Cw8 emerged as an independent risk factor for poor outcome (p = 0.03) following autologous transplantation in lymphoma patients. The association of HLA-Cw molecules with outcome in this study group indicates a need for further investigation of the HLA-mediated interactions that affect antitumor cytotoxicity, cytokine release, and regimen related toxicity.

A human immunoglobulin G receptor exists in both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms.

Several cDNA clones encoding the human immunoglobulin G receptor CD16 were isolated from human lung or peripheral blood leukocyte cDNA libraries. Nucleotide sequence comparisons revealed that the cDNAs could be divided into two groups. cDNA clones in one group encode a protein that terminates 4 amino acids after the putative transmembrane domain. Clones in the second group encode a protein with an extra 21 amino acids that could comprise a cytoplasmic domain. Direct peptide sequencing was used to determine the N terminus of the mature CD16 receptor protein and supported the existence of the two forms of the receptor. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C resulted in the release of a large percentage of the CD16 molecules from the cell surface. In contrast, treatment of natural killer cells with phosphatidylinositol-specific phospholipase C did not release any CD16 from the cell surface. These data demonstrate that both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms of the CD16 molecule exist and that they are differentially expressed on neutrophils and natural killer cells.