Immune parameters in patients with anxiety or depression during psychotherapy.

BACKGROUND: Numerous studies have described distinctive immunological findings in patients with depression. In contrast, only very little is known about the possible influence of anxiety disorders on the immune system. It is also unknown whether treatment with psychotherapy alone has any influence on immunological variations in patients with psychiatric disorders. METHODS: We measured immunological and psychological parameters in patients with minor depression (N=10) or anxiety disorder (N=13) over an 8-week course of inpatient psychotherapy. Data for patients and a group of healthy controls (N=11) were recorded three times in 4-week intervals. A FACS analysis revealed the composition of lymphocyte subpopulations. The production of reactive oxygen species (ROS) by phagocytes was analyzed using lucigenin-enhanced chemiluminescence. RESULTS: On admission, patients with anxiety disorder showed a markedly elevated ratio of CD4(+) (T helper) versus CD8(+) (T suppressor/cytotoxic) lymphocytes compared to healthy controls (P<0.001) and minor depressives (P<0.01). The increased ratio in patients with anxiety disorder could mainly be attributed to a reduced count in CD8(+) T cells compared to healthy controls (P<0.01) and depressives (P<0.05). There were no differences between patients with depression and healthy controls with respect to the CD4(+)/CD8(+) ratio. We did not observe any differences in the production of ROS by phagocytes in patients compared to healthy controls. The CD4(+)/CD8(+) ratio remained elevated in patients with anxiety disorders during the following 8 weeks. There were no significant changes in this parameter over the course of the inpatient treatment. LIMITATIONS: As a pilot study on the immune status in patients with anxiety disorders, the study's main limitation is the relatively low number of patients observed. CONCLUSIONS: In this study we demonstrated for the first time marked immunological changes in patients with anxiety disorders. In addition, our results provide preliminary evidence that these immunological variations are not reversible by an 8-week course of inpatient psychotherapy alone.

Acute psychological stress simultaneously alters hormone levels, recruitment of lymphocyte subsets, and production of reactive oxygen species.

Very little is known about the effects of acute psychological stress on the production of reactive oxygen species (ROS) by human phagocytic cells and the interplay between subjectively perceived stress, mediating hormones, variations in the number of peripheral leukocytes and ROS production. We measured psychological reactions, cardiovascular parameters, plasma catecholamines, plasma prolactin and cortisol as well as peripheral lymphocyte subsets in 13 experimental subjects undergoing a brief psychological stressor, and production of ROS, as indicated by chemiluminescence (CL), in stressed subjects and in healthy controls. The stressor elevated anger (p<0.01) and cardiovascular activation (p<0.01). There were significant changes in plasma levels of cortisol (p<0.01) and prolactin (p<0.001). During psychological stress natural killer (NK) cells (p<0.01) and CD8/CD38 cells (p<0.05) increased and returned to baseline only 25 minutes later. Significant changes in the number of naive CD4+/CD45RA+ (p<0.01) and antigen-experienced CD8+/CD45RO+ T cells (p<0.05) occurred. Subjects with stronger cardiovascular reaction showed higher stress-related plasma levels of norepinephrine (p<0.05) and were mainly responsible for the increase in NK cells. We observed a significantly reduced production of ROS following the stress test (p<0.05). Our results show that psychological stress is expressed simultaneously on psychological, hormonal and immunological levels of the organism. We show the existence of a circadian rhythm leading to a pronounced increase in CL during the morning hours. This first study taking this circadian rhythm in account revealed a significant suppressive effect of stress on ROS production.

Inverse relationship between patient peripheral blood CD34+ cell counts and collection efficiency for CD34+ cells in two automated leukapheresis systems.

BACKGROUND: The purpose of this study was to analyze the CD34 cell collection efficiency (CE) of automated leukapheresis protocols of two blood cell separators (Spectra, COBE [AutoPBSC protocol] and AS104, Fresenius [PBSC-Lym, protocol]) for peripheral blood progenitor cell (PBPC) harvest in patients with malignant diseases. STUDY DESIGN AND METHODS: PBPCs were collected by the Spectra AutoPBSC protocol in 95 patients (123 collections) and the AS104 PBSC-Lym protocol in 87 patients (115 harvests). Patients underwent a median of one (range, 1-4) conventional-volume apheresis procedure of 10.8 L (9.0-13.9) to obtain a target cell dose of > or =2.5 x 10(6) CD34+ cells per kg. RESULTS: The median overall CD34 CE was significantly better on the AS104 than on the Spectra: 55.8 percent versus 42.4 percent (p = 0.000). This was also true below (59.2% vs. 50.1%; p = 0.022) and above (51.2% vs. 41.3%; p = 0.001) the preleukapheresis threshold of 40 CD34+ cells per microL needed to collect a single-apheresis autograft. However, at > or =40 circulating CD34+ cells per microL, both cell separators achieved the target of > or =2.5 x 10(6) CD34+ cells per kg. The CD34 CE dropped significantly, from 59.2 percent at <40 cells per microL to 51.2 percent at > or =40 cells per microL on the AS104 (p = 0.017) and from 50.1 percent to 41.3 percent on the Spectra (p = 0.033). CONCLUSION: Whereas the CD34 CE was significantly different with the AS104 and the Spectra, the CD34 CE of both machines correlated inversely with peripheral blood CD34+ cell counts, showing a significant decline with increasing numbers of circulating CD34+ cells. Nevertheless, at > or 40 preapheresis CD34+ cells per microL, sufficient hematopoietic autografts of > or =2.5 x 10(6) CD34+ cells per kg were harvested by a single conventional-volume (11 L) leukapheresis on both cell separators.

Rituximab therapy of patients with B-cell chronic lymphocytic leukemia.

Rituximab (IDEC-C2B8) is a chimeric antibody that binds to the B-cell surface antigen CD20. Rituximab has significant activity in follicular non-Hodgkin lymphomas. Much less is known about the effects in chronic lymphocytic leukemia (CLL). We have initiated a phase II trial to evaluate the efficacy and safety of rituximab in patients with CD20+ pretreated CLL. To avoid the rituximab-associated toxicity, we restricted the tumor cell load, as measured by the number of circulating lymphocytes and the spleen size, in the first 2 cohorts of patients included in the study. Patients received 4 intravenous infusions of 375 mg/m2 once a week over a period of 1 month. Of the 28 patients evaluable for response, 7 patients showed a partial remission (National Cancer Institute criteria) lasting for a median of 20 weeks, with 1 patient still in remission after 71 weeks. Based on lymphocyte counts only, we found at least a 50% reduction of lymphocyte counts lasting for at least 4 weeks in 13 (45%) of 29 patients. Fifteen patients from 3 institutions were monitored for the immunophenotype profile of lymphocyte subsets. The number of CD5+CD20+ cells decreased significantly and remained low until day 28 after therapy. T-cell counts were not affected. With the exception of one rituximab-related death, adverse events in the remaining patients were mild. The results suggest that rituximab has clinical activity in pretreated patients with B-CLL. Toxicity is tolerable. Response duration after withdrawal of rituximab is rather short. Therefore, other modes of application and the combination with other agents need to be tested.

Monoclonal antibody FMC7 detects a conformational epitope on the CD20 molecule: evidence from phenotyping after rituxan therapy and transfectant cell analyses.

Numerous studies have reported that monoclonal antibody (mAb) FMC7 detects an antigen present on only a subset of circulating B lymphocytes. In particular, this mAb may distinguish typical B-cell chronic lymphocytic leukemia (FMC7 negative) from other types of B-cell non-Hodgkin lymphoma (B-NHL; FMC7 positive). We treated patients with B-NHL with Rituxan, a chimeric CD20 mAb, and observed abrogation of staining not only with prototype CD20 mAb B-1 but also with mAb FMC7. To investigate the relation between antigens CD20 and FMC7, we performed mutual blocking studies that showed mutual inhibition of FMC7 and CD20. In addition, FMC7 modulated CD23 expression and confirmed the presence of mAb B-1 in B-lymphoblastoid cell lines CESS and JVM. Transient transfection of myeloid cell line K562 with plasmid containing CD20-encoding cDNA produced de novo expressions of CD20 and FMC7. Our data indicate that FMC7 binds to a particular conformation of the CD20 antigen, probably to a multimeric CD20 complex. We assume that FMC7 stains positively only when CD20 antigen is present in high densities and in the postulated multimeric complex formation.

A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts.

Recently, the regulatory authorities have begun to show interest in haematopoietic stem cell products. On a professional rather than a regulatory basis, the International Society for Hematotherapy and Graft Engineering (ISHAGE) has established the Foundation for the Accreditation of Haematopoietic Cell Therapy (FACHT), which has drawn up guidelines for standards and accreditation of such activity. In Europe, the regulatory environment with regard to haematopoietic stem cell grafts, processing and storage are currently less stringent. However, in 1998 the European Joint Accreditation Committee Euro-ISHAGE/EBMT (JACIE) prepared a regulatory document 'Standards for Blood and Marrow Progenitor Cell Collection, Processing and Transplantation' which was approved by the EBMT General Assembly. The major objectives were to promote quality of medical and laboratory practice in haematopoietic progenitor cell transplantation. The standards extend and detail the pre-existing activity of EBMT centres including all phases of collection, processing and administration of these cells. This is the platform for the proposed reference protocol for CD34(+) cell enumeration and clinical validation of quality assessment to ensure that appropriate standards of work and product quality are established and will be maintained.

PBPC mobilization with paclitaxel, ifosfamide, and G-CSF with or without amifostine: results of a prospective randomized trial.

BACKGROUND: The impact of amifostine on PBPC mobilization with paclitaxel and ifosfamide plus G-CSF was assessed. STUDY DESIGN AND METHODS: Forty patients with a median age of 34 years (range, 19-53) who had germ cell tumor were evaluated for high-dose chemotherapy. Patients were randomly assigned to receive either a single 500-mg dose of amifostine (Group A, n = 20) or no amifostine (Group B, n = 20) before mobilization chemotherapy with paclitaxel (175 mg/m(2)) given over 3 hours and ifosfamide (5 g/m(2)) given over 24 hours (TI) on Day 1. G-CSF at 10 microg per kg per day was given subsequent to TI with or without amifostine from Day 3 until the end of leukapheresis procedures. RESULTS: In 2 (10%) of 20 patients receiving amifostine and 3 (15%) of 20 patients not receiving it, no PBPC separation was performed because of mobilization failure. No significant differences were observed in the study arms with regard to the time from chemotherapy until first PBPC collection or the number of apheresis procedures needed to harvest more than 2.5 x 10(6) CD34+ cells per kg. Furthermore, leukapheresis procedures yielded comparable doses of CD34+ cells per kg (3.4 x 10(6) vs. 3.6 x 10(6); p = 0.82), MNCs per kg (2.7 x 10(8) vs. 2.6 x 10(8); p = 0.18), and CFU-GM per kg (15.9 x 10(4) vs. 19.3 x 10(4); p = 0.20). Patients in Group A had higher numbers of circulating CD34+ cells on Day 10 (103.0/microL vs. 46.8/microL; p = 0.10) and on Day 11 (63.0/microL vs.14.3/microL; p = 0.04) than did patients in Group B. CONCLUSION: Administration of a single dose of amifostine before chemotherapy with TI mobilized higher numbers of CD34 cells in the circulation, but did not enhance the overall collection efficiency in the present trial.

Automated collection of peripheral blood stem cells with the COBE spectra for autotransplantation.

BACKGROUND: A convenient, effective and safe peripheral blood stem cell (PBSC) apheresis procedure is desirable to cope with the increasing requirements for PBSC collections. We performed PBSC harvesting with the novel COBE Spectra AutoPBSC(TM) system using the default software configuration recommended by the manufacturer. We analyzed collection parameters and clinical efficiency of harvested autografts following high-dose chemotherapy (HDCT). PATIENTS AND METHODS: Eighty-one patients underwent 102 harvests after standard chemotherapy plus filgrastim (5-10 microg/kg/day) to obtain a target of >/=2.5 x 10(6) CD34+ cells/kg for autologous blood stem cell transplantation. Conventional-volume leukaphereses (median: 11 liters) were performed using the manufacturer's standard software default regarding inlet flow, harvest/chase volume (3/7 ml) and number of collection cycles. The ratio of ACD-A to whole blood was initially set at 1:12 (56 collections), later at 1:10 (46 aphereses). RESULTS: With respect to preapheresis counts of 93 (9-876) CD34+ cells/microl, 69 patients (85.2%) achieved >/=2.5 x 10(6) CD34+ cells/kg by the first apheresis. PBSC products contained medians of 5.0 x 10(6) (0.7-77.3) CD34+ cells/kg and 13.8 x 10(4) (2.3-105.0) CFU-GM/kg. A preapheresis count of >/=40 CD34+ cells/microl predicted a single-apheresis yield of >/=2.5 x 10(6) CD34+ cells/kg. Apheresis products showed a high mononuclear cell (MNC) purity of >/=89%. The median overall collection efficiency of CD34+ cells (CD34-CE) was 42.6% (12.2-87.4). The CD34-CE decreased significantly with increasing numbers of circulating CD34+ cells: 52.5% at CD34+ cells <40/microl versus 41.0% at CD34+ cells >/=40/microl (p 0.5 x 10(3)/microl, 10 (8-13) days for WBC >1.0 x 10(3)/microl and 11 (8-17) days for platelets >20 x 10(3)/microl. CONCLUSIONS: As a result of efficient PBSC mobilization, a single conventional-volume leukapheresis with the COBE Spectra AutoPBSC system resulted in hematopoietic autografts with >/=2.5 x 10(6) CD34+ cells/kg in 85% of patients. Following the standard PBSC apheresis recommendations of the manufacturer, the AutoPBSC system assures PBSC products with a high MNC purity and a moderate CD34-CE that declines significantly at increasing levels of circulating CD34+ cells. Leukaphereses performed at an ACD-A to whole blood ratio of 1:10 should run without coagulation problems.

Circulating CD4+ T lymphocytes with intracellular but no surface CD3 antigen in five of seven patients consecutively diagnosed with angioimmunoblastic T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL) accounts for less than 1% of all lymphatic malignancies. Oligoclonality or monoclonality for any of the T-cell receptor (TCR) chain genes can be demonstrated in the majority of the cases. During systematic screening for the presence of circulating lymphocytes with atypical coexpression of differentiation antigens in patients with T-cell lymphomas, we have discovered a minor population (accounting for 0.2% to 10.% of all lymphocytes) of atypical lymphocytes in the blood of five of seven patients consecutively diagnosed in 1997/1998 by lymph node histology to have AITL. The major distinguishing feature of these cells consists of the lack of the surface expression of the CD3 antigen, but not of the intracellular expression. These cells express the T-cell antigens CD2 and CD5 on their surface, but not CD7, and they express CD4 and CD45 at numbers of molecules per cell typical for T lymphocytes. Gene scan analyses for the TCR gamma chain revealed oligoclonality of these flow-sorted cells in one patient and monoclonality in two patients, the same patterns of TCR gamma chain gene as determined processing the respective diagnostic lymph nodes. Circulating CD4-expressing T lymphocytes with exclusively cytoplasmic expression of CD3 appear to represent the malignant population in patients with histologically diagnosed AITL.

Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield.

BACKGROUND: In immunomagnetic selection of CD34+ cells from HPC transplants, not all factors that affect yield and purity of CD34+ cells are known. METHODS: Forty-three consecutive procedures of immunomagnetic selection of CD34+ cells from peripheral blood HPCs and bone marrow harvests (autologous harvests, n = 27; allogeneic harvests; n=16) were performed by use of a cell selection system (Isolex 300i, Baxter Immunotherapy). The composition of the starting component and the subsets of CD34+ cells were analyzed for correlation with the yield and purity of the final component. RESULTS: The mean purity of the final components was 84.3 percent (range, 27-99%), and the mean yield was 51.4 percent (range, 9.4-80. 4%). Partial regression analysis showed that, among the factors correlating with purity and/or yield, the RBC volume in the starting fraction had the highest predictive impact on the purity and yield of CD34+ cells, even after the exclusion of procedures using bone marrow harvests as an HPC source (beta coefficient, -0.704; p = 0. 001). CONCLUSION: The use of the Isolex 300i system allows efficient recovery of CD34+ cells in routine selection procedures. The volume of RBCs in the starting component should be minimized to ensure a high yield and purity of the final component.

Elevated reticulocyte count--a clue to the diagnosis of haemolytic-uraemic syndrome (HUS) associated with gemcitabine therapy for metastatic duodenal papillary carcinoma: a case report.

In adults, the haemolytic-uraemic syndrome (HUS) is associated with probable causative factors in the minority of all cases. Cytotoxic drugs are one of these potential causative agents. Although metastatic cancer by itself is a recognized risk-factor for the development of HUS, therapy with mitomycin-C, with cis-platinum, and with bleomycin carries a significant, albeit extremely small, risk for the development of HUS, compared with all other cytotoxic drugs. Gemcitabine is a novel cytotoxic drug with promising activity against pancreatic adenocarcinoma. We are reporting on one patient with metastatic duodenal papillary carcinoma developing HUS while on weekly gemcitabine therapy. The presenting features in this patient were non-cardiac pulmonary oedema, renal failure, thrombocytopenia and haemolytic anaemia. The diagnosis of HUS was made on the day of admission of the patient to this institution. Upon aggressive therapy, including one single haemodialysis and five plasmaphereses, the patient recovered uneventfully, with modestly elevated creatinine-values as a remnant of the acute illness. Re-exposure to gemcitabine 6 months after the episode of HUS instituted for progressive carcinoma, thus far has not caused another episode of HUS.

Influenza vaccination in liver transplant recipients.

BACKGROUND: The immunogenicity of the trivalent inactivated influenza split virus vaccine (Infusplit SSW 97/98) containing A/Bayern/07/95 (H1N1)-like (A/Johannesburg/82/96 [NIB-39]), A/Wuhan/359/95 (H3N2)-like (A/Nanchang/933/95 [Resvir-0]), and B/Beijing/184/93-like (B/Harbin/7/94) hemagglutinin antigens was tested in liver transplant recipients (TXL-R). SUBJECTS AND METHODS: Serum antibody titers were determined 21+/-2 days after a single vaccination in 62 adult TXL-R and 59 adult volunteers. RESULTS: Protective postimmunization antibody titers for the three antigens were similar in TXL-R (protection rates 92%, 92%, and 95%) and the comparison group (97%, 100%, and 100%). Adverse reactions were mild and less frequent in TXL-R. A significant decrease of CD8+CD38+ lymphocytes after vaccination was found in TXL-R. No association between antibody response and age, gender, time interval since transplantation, anti-hepatitis B surface antigen immunoprophylaxis, or immunosuppressive medication was detected. CONCLUSION: Our results show that the vaccine is safe and effective and should be recommended to TXL-R.

Important role of nondiagnostic blood loss and blunted erythropoietic response in the anemia of medical intensive care patients.

OBJECTIVE: To determine incidence, severity, characteristics, and causes of anemia and transfusion requirements in medical intensive care patients. DESIGN AND SETTING: Open prospective clinical study in a 24-bed medical intensive care unit in a tertiary-care university hospital. PATIENTS: Patients (N = 96) treated in the intensive care unit for >3 days. INTERVENTIONS: None. MEASUREMENTS: Parameters of erythropoiesis and red blood cell metabolism, including hemoglobin, reticulocyte counts, serum iron, transferrin, ferritin, haptoglobin, vitamin B12, folic acid, and erythropoietin concentrations were determined serially. Diagnostic blood loss and red blood cell transfusions were recorded, and the total blood loss was estimated from changes in hemoglobin concentrations and the amount of hemoglobin transfused. MAIN RESULTS: The median hemoglobin concentration was 12.1 g/dL at admission and 11.2 g/dL at the end of the intensive care unit stay. A total of 74 patients (77%) suffered from anemia and received 257 red blood cell units, approximately half of which were given within the first 5 days. Three patients who received 19 red blood cell units were admitted with acute gastrointestinal bleeding, but in the remainder, a median total blood loss of 128 mL/d was not (n = 60) or not solely (n = 11) a result of overt bleeding. Diagnostic blood loss declined from a median of 41 mL on day 1 to <20 mL after 3 wks and contributed 17% (median) to total blood loss. Acute renal failure, fatal outcome, and simplified acute physiology score >38 on admission were associated with a 5.8-, 7.0-, and 2.8-fold increase in total blood loss. Reticulocyte counts and erythropoietin concentrations were inappropriately low for the degree of anemia, and plasma transferrin saturation was mostly <20%. CONCLUSIONS: Anemia is frequent and results in a high requirement for red blood cell transfusions in the medical intensive care setting. A major proportion of blood loss is not caused by overt bleeding or diagnostic blood sampling but, rather, may result from various other reasons, e.g., occult gastrointestinal bleeding and renal replacement therapy. The erythropoietic response to anemia is blunted, probably as a consequence of an inappropriate increase in erythropoietin production and diminished iron availability. (Crit Care Med 1999; 27:2630-2639)

Quantitative fluorescence flow cytometry: a comparison of the three techniques for direct and indirect immunofluorescence.

Three types of microbead calibrators available for quantitative fluorescence flow cytometry have been studied in parallel using a variety of monoclonal antibodies (MoAbs). The QIFI kit is designed for indirect immunofluorescence (IF), and both the Quantum Simply Cellular (QSC) assay and the Quanti-BRITE assay are designed for direct IF. Because of the different nature of the respective ligands, epitopes on cells versus F'ab-portions on QSC beads, large differences in titration curves for a large number of CD MoAbs were noted between QSC beads and cells. Use of the QSC assay and fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugates of the same CD reagent revealed substantially different numbers of cellular binding sites. Numbers of cellular binding sites as determined by direct IF using the Quanti-BRITE assay and by indirect IF using the QIFI kit were similar. We also found that erythrocyte (RBC)-lysing reagents cause varying and sometimes substantial reduction in the fluorescence intensity (FI) of cells stained directly with CD34 MoAb conjugates, but the RBC-lysing reagents had no effect on the FI of cells stained indirectly with the same CD34 MoAbs. This report defines a number of variables critical for standardized quantitative flow cytometry. We conclude that the choice of calibrators, fluorochrome conjugates, staining methods, and modes of sample processing can effect the determination of cellular binding sites to MoAbs. Direct immunofluorescence using the Quanti-BRITE assay and indirect IF using the QIFI kit appear to yield comparable results for the standardized determination of numbers of cellular binding sites to MoAbs.