Clinical, microbiological, and immunological characteristics in HIV-infected subjects at risk for disseminated Mycobacterium avium complex disease: an AACTG study

The clinical, microbiologic, and immunologic parameters in HIV-infected subjects first presenting with disseminated Mycobacterium avium complex (DMAC) were determined. Four HIV-positive groups not yet on DMAC treatment were enrolled: 19 subjects with CD4 lymphocyte counts < or =50/microl thought to have DMAC on clinical grounds; 18 subjects newly found to have a positive blood culture for MAC; 25 asymptomatic controls (CD4 cell counts < or =50); and 25 asymptomatic controls (CD4 counts 100-250/microl). Outcome measures include comparisons between groups for clinical characteristics; results of cultures from blood, marrow, and gastrointestinal and respiratory tracts; immunological markers from staining of marrow and flow cytometry of circulating lymphocytes; and cytokine production of PBMCs. Only 21% of the 19 patients entered on suspicion of having DMAC grew MAC from blood or marrow. Neither clinical presentation nor laboratory tests differentiated those culture-positive from those culture-negative patients. However, prior PCP or multiple other opportunistic infections were more common in the DMAC group. MAC was isolated from 82% of marrow and 50% of blood specimens from the DMAC group. Respiratory or gastrointestinal colonization was present in 36% of DMAC subjects, but only 5% of non-DMAC subjects with CD4 counts <50 cells/microl. CD8+ cells were more frequent in bone marrow, and CD4 cells recognizing MAC antigen were more frequent in blood from DMAC subjects vs. controls. Results suggest an early stage of tissue dissemination preceding persistent bacteremia, and mucosal entry without persistence of colonization. MAC-specific T cell responses apparently develop and persist during DMAC, but are dysfunctional or too infrequent to prevent persistence.     

The in vitro effect of leucocyte alpha-interferon on human myeloma cells in a semisolid agar culture system

73 bone marrow samples from a total of 50 patients with multiple myeloma (MM) were tested for interferon (IFN) sensitivity in the human tumour colony assay (HTCA). 16 evaluable samples were obtained from untreated patients, 10 from patients during melphalan and prednisone treatment and 6 from patients on IFN treatment or after withdrawal of IFN. The sensitivity to IFN was individually distributed between the bone marrow samples from patients of all 3 groups and a tendency towards IFN resistance could be found in serial assays from patients during MP-treatment and after IFN withdrawal. Stimulation of growth was found in 31% of the cultures, usually at lower doses of IFN (10-100 U/ml) while higher doses (400-4000 U/ml) inhibited growth in 75% of the assays.     

The in vitro effect of leucocyte α-interferon on human myeloma cells in a semisolid agar culture system

73 bone marrow samples from a total of 50 patients with multiple myeloma (MM) were tested for interferon (IFN) sensitivity in the human tumour colony assay (HTCA). 16 evaluable samples were obtained from untreated patients, 10 from patients during melphalan and prednisone treatment and 6 from patients on IFN treatment or after withdrawal of IFN. The sensitivity to IFN was individually distributed between the bone marrow samples from patients of alle 3 groups and a tendency towards IFN resistance could be found in serial assays from patients during MP-treatment and after IFN withdrawal. Stimulation of growth was found in 31% of the cultures, usually at lower doses of IFN (10–100 U/ml) while higher doses (400–4000 U/ml) inhibited growth in 75% of the assays.     

Evaluation of viral load and antigenemia as markers for relapse cytomegalovirus infection in renal transplant recipients

Cytomegalovirus (CMV) is a pathogen, commonly found in the donors and recipients of solid organ transplantation. CMV is one of the major causes of morbidity and mortality in these patients. Relapsing episodes of CMV infection occur in 23-33% of transplant patients which is likely a reflection of incomplete suppression of viral replication following antiviral treatment with intravenous ganciclovir. We have studied CMV DNA load and antigenemia as markers for relapse of CMV infection in 49 renal transplant patients out of 68 with CMV infection who received a course of intravenous ganciclovir among 300 transplants carried out between January of 2001 and June of 2005. Viral load and antigenemia were measured in blood samples obtained before, during and at the completion of treatment. We also studied different viral load as predictors of relapse CMV infection. Twelve (24.5%) of 49 recipients developed relapsing CMV infection. The relapsing group had higher viral loads after treatment than the no relapsing group. There was no difference in antigenemia level between both groups. The viral loads before and during the treatment, the age and sex of donors and recipients, inmunosupresión, percentage of seronegative recipients with seropositive donors, duration of the therapy and the percentage of patients with heavy immunosuppression were similar in the two groups, but the incidence of acute rejection was higher in the relapsing group. We also evaluated the range of viral load after treatment which is able to trigger the relapse of CMV infection. We conclude that CMV DNA load after treatment is a useful marker for individualizing antiviral treatment of CMV infection in renal transplant recipients. Acute rejection is a risk factor to the relapsing CMV infection.     

Studies of hemopoietic stromal fibroblastic colonies in patients undergoing bone marrow transplantation

We report here the results of serial bone marrow fibroblast cultures from recipients of histocompatible allogeneic bone marrow transplants (37 patients with acute leukemia, and seven with severe aplastic anemia). The mean value of marrow CFU-F growth for recipients at day 21 after transplant was significantly lower than that for normals and for patients before transplant. There were no obvious differences in the morphologic, ultrastructural, and cytochemical characteristics of the CFU-F between the normals and the patients who received transplants. The number of stromal CFU-F cells in S phase was increased during the early period after transplantation, as demonstrated by a significant reduction of CFU-F growth after short exposure to hydroxyurea. However, the return to normal was rapid, within six weeks. The presence of graft-vs-host disease (greater than or equal to grade II) and the choice of immunosuppressive drug, i.e., methotrexate or cyclosporine, did not affect the CFU-F recovery. The findings in the present study show that the marrow stromal compartment is compromised after marrow transplantation, but its regeneration is rapid. No in vitro CFU-F growth was obtained from peripheral blood of donors and patients, either before or after bone marrow transplant.     

Impaired in-vitro growth of megakaryocytic colonies derived from CD34 cells of HIV-1-infected patients with active viral replication

OBJECTIVE: To address the mechanisms of the thrombocytopoietic dysfunction that may follow HIV infection and to compare peripheral blood and bone marrow as sources of CD34 progenitor cells in HIV-infected patients. METHODS: The study used CD34 progenitor cells from 20 previously untreated HIV-infected individuals, 20 HIV-infected individuals treated with antiretroviral therapy and a control group of 20 HIV-uninfected healthy individuals to examine in-vitro megakaryocytopoiesis. There were no hematological abnormalities at baseline in the study groups. CD34 progenitor cells derived from peripheral blood and bone marrow were purified and cultured in medium containing thrombopoietin, interleukin-3, and interleukin-6. HIV-1 plasma viral load was determined by b-DNA technique. Expression of receptors for thrombopoietin, interleukin-3, and interleukin-6 was assessed on CD34 cells by flow cytometry, and numbers of receptors per single cell were calculated by Quanticalc software. RESULTS: Growth of megakaryocytopoietic colony-forming units (CFU-MK) were impaired in untreated HIV-infected individuals despite normal platelet counts. Viral load levels inversely correlate with CFU-MK growth and platelet counts. Antiretroviral drug-treated individuals showed normal megakaryocyte development. Similar results were obtained whether the CD34 progenitor cells derived from peripheral blood or bone marrow. CONCLUSIONS: These findings suggest that megakaryocyte differentiation is impaired before the onset of overt thrombocytopenia in HIV-infected patients and provide evidence for a direct link between viral replication and perturbed megakaryocytopoiesis, which appears to be prevented and/or restored by antiretroviral therapy. The results indicate that peripheral blood represents a suitable source of CD34 hematopoietic progenitors for studies of megakaryocytopoiesis in HIV disease.     

Lung function 5 yrs after allogeneic bone marrow transplantation conditioned with busulphan and cyclophosphamide.

Long-term data on lung function after bone marrow transplantation (BMT) are inconclusive. Previously, a persistent reduction in gas transfer 1 yr after allogeneic BMT with busulphan and cyclophosphamide conditioning was reported by the current authors. In the present study this reduction was examined to see if it was permanent, transient or progressive. Prospectively, 43 consecutive adult patients with malignant blood disorders undertook lung function measurements prior to BMT, at 3 month intervals during the 1st yr after BMT and finally after 5 yrs. Mean baseline lung function values were >90% predicted. Within the 1st yr after BMT a transient decline in lung volumes and a persistent reduction in gas transfer were observed. After 5 yrs, baseline values were restored for all variables, except in four patients who developed obliterative bronchiolitis. Acute leukaemia and smoking were independently associated with gas transfer reductions at baseline and during the 1st yr after BMT. Allogeneic bone marrow transplantation with busulphan and cyclophosphamide conditioning was associated with a reduction in gas transfer 1 yr after bone marrow transplantation but baseline values were usually restored after 5 yrs. Since recovery may be gradual and slow, an observation period >1 yr is required before drawing conclusions concerning the development of a permanent reduction in lung function after allogeneic bone marrow transplantation conditioned with busulphan and cyclophosphamide.     

Tumor load in patients with follicular lymphoma post stem cell transplantation may correlate with clinical course

The purpose of this study was to evaluate if the tumor load, as determined by a real-time quantitative PCR (RQ-PCR) assay, correlated with the clinical course of follicular lymphoma patients after stem cell transplantation (SCT). Cryopreserved bone marrow and/or peripheral blood samples obtained at different time intervals after SCT from 11 patients (seven allogeneic, T-cell depleted/four autologous) were tested for tumor load, as defined by t(14;18) positive cells/total cells, using RQ-PCR. None of the six patients who remained in remission had samples with a tumor load >0.01% after SCT, although fluctuating tumor loads of 0.01% after SCT (0/6 vs 4/5, P<0.02, Fisher's exact). Our results suggest that RQ-PCR measurable tumor load >0.01% after SCT may correlate with relapsed/progressive disease. Prospective studies with greater numbers of cases are indicated to better determine the critical tumor load that predicts poor outcome after SCT with RQ-PCR.     

Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia

In children with acute lymphoblastic leukemia (ALL), response to treatment is assessed by bone marrow aspiration. We investigated whether minimal residual disease (MRD) can be effectively monitored in peripheral blood. We used flow cytometric techniques capable of detecting 1 leukemic cell among 10 000 or more normal cells to compare MRD measurements in 718 pairs of bone marrow and peripheral blood samples collected from 226 children during treatment for newly diagnosed ALL. MRD was detected in marrow and blood in 72 pairs and in marrow but not in blood in 67 pairs; it was undetectable in the remaining 579 pairs. Remarkably, findings in marrow and blood were completely concordant in the 150 paired samples from patients with T-lineage ALL: for each of the 35 positive marrow samples, the corresponding blood sample was positive. In B-lineage ALL, however, only 37 of 104 positive marrow samples had a corresponding positive blood sample. Notably, peripheral blood MRD in these patients was associated with a very high risk for disease recurrence. The 4-year cumulative incidence of relapse in patients with B-lineage ALL was 80.0% +/- 24.9% for those who had peripheral blood MRD at the end of remission induction therapy but only 13.3% +/- 9.1% for those with MRD confined to the marrow (P =.007). These results indicate that peripheral blood may be used to monitor MRD in patients with T-lineage ALL and that peripheral blood MRD may provide strong prognostic information in patients with B-lineage ALL.     

Morphology of cells grown in the CFU-GEMM tissue culture assay from mononuclear cells obtained from peripheral blood and bone marrow of normal volunteers

In ten healthy volunteers studies were done to assess the morphology, immunocytology, and cytochemical properties of mononuclear hematopoietic stem cells isolated from normal human peripheral blood and bone marrow grown in semisolid matrix mixed lineage colony-forming unit (CFU-GEMM) culture. In three volunteers, peripheral blood and bone marrow samples were collected simultaneously; the progeny were remarkably similar in each of the three paired cultures, although macrophages were more numerous in the bone marrow cultures. Three peripheral blood samples were cultured following frozen storage with 10% DMSO at-150 degrees C for approximately 5 months. Megakaryopoiesis was present in each case, demonstrating the full hematopoietic potential of previously frozen peripheral blood mononuclear cells. Four other peripheral blood samples and one bone marrow sample were cultured and, in each case, cells of all hematopoietic cell lines were present. Bone marrow, peripheral blood mononuclear cells, and previously frozen peripheral blood mononuclear cells grown in the CFU-GEMM tissue culture assay showed the presence of granulocyte-macrophage, erythroid, and megakaryocytic cell lines.     

Influence of peripheral blood admixture on the number of hematopoietic progenitor cells (CFU-GM and BFU-E) in human bone marrow aspirates

Peripheral blood contamination in human bone marrow aspirates was calculated from the ratios between the erythrocyte and nucleated cell counts in both the bone marrow aspirate and a simultaneously obtained peripheral blood sample. This method is based upon experimental data which showed that the erythrocytes in bone marrow aspirates are mainly derived from the intravascular blood compartment. A strong negative correlation was found between the peripheral nucleated cell fraction (FBl) and the number of myeloid progenitor cells in 65 bone marrow samples (correlation coefficient r = -0.51; p less than 0.001). A similar correlation was found between erythroid progenitor cells and peripheral blood fraction (r = -0.55; p less than 0.01). The culture conditions were continuously monitored, using large batches of frozen marrow samples as controls. The correction for peripheral blood admixture permits a more reliable and reproducible interpretation of the quantitative results obtained from studies on the number of clonogenic cells in human bone marrow aspirates. Moreover, the method allows the interpretation of data obtained from bone marrow samples which are heavily contaminated by peripheral blood.     

Improvement in ventricular function during exercise studied with radionuclide ventriculography after cardiac rehabilitation

A heterogeneous group of 19 consecutive patients with coronary artery disease were studied with radionuclide ventriculography before and after a mean of 6 months of exercise training. Ejection fraction was measured at rest, at matched submaximal supine work loads and during maximal supine bicycle exercise. After training there was no change in mean ejection fraction at rest or during maximal exercise, but a higher maximal mean systolic blood pressure, heart rate and work load were achieved. At equivalent submaximal work loads after training, similar levels of mean heart rate and systolic blood pressure were reached but a statistically greater mean ejection fraction was obtained. These preliminary results suggest that exercise training may improve cardiac function during exercise in selected patients with coronary disease. A randomized study using similar techniques has been initiated.     

Lymphocyte subsets in normal human bone marrow harvested for routine clinical transplantation

Bone marrow and peripheral blood of 25 healthy bone marrow donors from our allogeneic bone marrow transplantation program were assessed for cell subsets bearing T11(CD2), T4(CD4), T8(CD8), B1(CD20) J5(CALLA, CD10), Mo1(CD11b), MY7(CD13). Mo2(CD14), MY9(CD33) and NKH-1 antigens. Bone marrow cell samples were taken for analysis at the start or at the end of the harvesting procedure of aspiration from the iliac crest. All samples were analysed on a flow cytometer at the lymphocyte window as obtained on the two-parameter (L90oLSxFALS) scatter diagram. There were no differences in the lymphocyte subset composition of bone marrow samples taken at the start or at the end of the harvesting procedure. In contrast to the majority of literature data, a high CD4/CD8 ratio was detected in bone marrow samples: it did not differ from that in the peripheral blood. The proportions of CD2 and CD4 T cell markers in the bone marrow correlated with those in the peripheral blood, thus further documenting a substantial bone marrow contamination with peripheral blood cells. A relatively large aspirate volume (4-5 ml) obtained from individual aspiration sites was identified as the only factor possibly accounting for the high-level contamination of bone marrow samples with peripheral blood. This conclusion was corroborated by low T cell proportions and low CD4/CD8 ratios found in the bone marrow washed from bone fragments and in bone marrow samples aspirated at first bone puncture in a volume of 1.0 ml. Taken together, these findings imply that less vigorous suction may decrease the number of T lymphocytes in bone marrow harvested for transplantation purposes.     

Contamination during in vitro processing of bone marrow for transplantation: clinical significance

We determined the prevalence and clinical significance of positive microbiologic cultures obtained from 194 consecutive bone marrow harvests intended for infusion in 188 consecutive adult bone marrow transplant patients. Microbiologic cultures were obtained at harvest, after manipulation in vitro (for ABO imcompatibility or purging procedures), and at the time of thawing and infusion (after earlier cryopreservation). Only one of 194 marrow harvests was culture-positive intra-operatively (from an ABO-compatible allogeneic marrow that was infused without manipulation). None of 39 other allogeneic marrows (including 21 ABO-incompatible marrows which were manipulated in vitro) and none of 154 autologous marrows (including 40 which were purged in vitro) grew bacteria or fungi. On the other hand, 12 of 153 (8%) bone marrow samples were positive for micro-organisms after thawing at the time of infusion. The predominant organisms cultured were gram negative bacilli (including five Pseudomonas sp.), probably introduced during the thawing process in a water bath. In only one of 13 contaminated marrows was the same organism(s) (Pseudomonas picketti and Pseudomonas paucimobilis) recovered in vivo during the transplant course. This patient experienced a bacteremia which was eradicated without sequelae. Contamination of marrow can occur during the procurement and in vitro handling processes. With proper sterile technique bone marrow infusion does not pose a significant infectious risk for the immunocompromised transplant patient.     

Bedside leukoreduction of cellular blood components in preventing cytomegalovirus transmission in allogeneic bone marrow transplant recipients: a retrospective study.

BACKGROUND AND OBJECTIVES: Cytomegalovirus (CMV) infection continues to be a major complication of bone marrow transplants (BMTs). Administration of leukoreduced unscreened cellular blood products at the bedside has been shown to be effective in preventing CMV transmission via transfusions in CMV-seronegative bone marrow transplant recipients who receive their transplants from CMV-seronegative donors. The aim of this study was to determine whether CMV infection occurred in CMV-seronegative BMT patients who received CMV-seronegative donor marrows and CMV untested blood products leukodepleted at the bedside. DESIGN AND METHODS: We collected data over a 2-year period from patients undergoing allogeneic transplantation who received leukoreduced cellular blood components that were not screened for CMV. All CMV-seropositive patients and donors were excluded from the study. The CMV status of both the donors and the patients was determined before the transplantations. CMV cultures of urine, blood buffy coat, bone marrow samples and bronchial washings were performed if necessary in patients. RESULTS: Thirty-six CMV-seronegative patient-donor pairs were included in the study. Five patients (13.89%) were serologically reactive, but their CMV cultures were negative and they did not show signs or symptoms of CMV infection. These patients received intravenous immunoglobulin and thus could have acquired anti-CMV passively. INTERPRETATION AND CONCLUSIONS: The confidence interval in this study is 0/36 incidence of CMV infection. Our present findings support those of prior studies showing the effectiveness of filtered unscreened blood components as an alternative transfusion support for CMV-seronegative marrow transplant recipients. Studies in larger number of patients are warranted.     

Speed of relapse in children with acute lymphoblastic leukaemia

16 children with acute lymphoblastic leukaemia (ALL) who relapsed on maintenance treatment between September 1978 and December 1981 were studied. Their previous bone marrow aspirates were reviewed to determine the first evidence for marrow relapse and the subsequent rate of evolution of the disease. 60% of children had bone marrow evidence of unsuspected relapse at 8 wk, and 40% had evidence at 12 wk before clinical or peripheral blood relapse occurred. Early detection of relapse will prevent continued use of ineffective maintenance chemotherapy and may also reduce morbidity during subsequent induction therapy. Regular 8-weekly marrow aspirates are therefore recommended during first remission of ALL in children with a histocompatible sibling who would be eligible for bone marrow transplantation in second remission.     

Increased granulocytic, erythrocytic, and megakaryocytic progenitors in myelofibrosis with myeloid metaplasia

Nucleated cells obtained from blood and/or bone marrow of patients with myelofibrosis with myeloid metaplasia (MMM) were cultured in diffusion chambers (DC) implanted into the peritoneal cavities of irradiated mice. A total of five blood studies and two bone marrow studies were performed using cells obtained from five patients. The DC were harvested at intervals from the host mice and the total and differential cellularity of DC contents were evaluated. The results obtained from MMM cultures were compared with those from similar cultures of blood cells and marrow cells of four and six normal individuals respectively. The proliferation and maturation of the granulocytic, erythrocytic, and megakaryocytic lines in MMM cultures occurred in an orderly fashion as they occur in vivo. The patterns of proliferation and maturation of the three cell lines in cultures after day 7 suggest that they primarily originate from progenitor cells. The numbers of granulocytes in the multiplicative pool, recognizable red cell precursors, and megakaryocytes recovered were significantly greater from the MMM cultures than those from the normal blood or marrow cultures. These results suggest that the blood and marrow cells of MMM patients have increased numbers of progenitors for granulocytes, erythrocytes, and megakaryocytes.     

The effects of intranasal budesonide on allergen-induced production of interleukin-5 and eotaxin,airways, blood,and bone marrow eosinophilia,and eosinophil progenitor expansion in sensitized mice

We have previously demonstrated that allergen inhalation induces expansion of bone marrow eosinophil progenitors in sensitized mice and subjects with asthma and that the inhaled corticosteroid, budesonide, reduced baseline but not allergen-induced increase in bone marrow eosinophil/basophil progenitors (EoB-CFU) in subjects with asthma. Here, we evaluated the effects of intranasal budesonide on allergen-induced increases in interleukin (IL)-5 and eotaxin in the airway and peripheral blood, expansion of bone marrow Eo-CFU and eosinophilia in bone marrow, peripheral blood and airway, as well as airway hyperresponsiveness, in ovalbumin (OVA)-sensitized mice. Budesonide treatment attenuated allergen-induced eosinophilia in bone marrow, peripheral blood, and airways as well as allergen-induced increases in bone marrow eosinophil progenitors but not allergen-induced increases in IL-5 or eotaxin 12 h following the second of two daily exposures to allergen; at later time points treatment was associated with attenuation of IL-5, eosinophilia, Eo-CFU, and airway hyperresponsiveness. These results suggest that a component of the mechanism by which corticosteroid treatment attenuates allergen-induced airway inflammation is through suppression of bone marrow eosinophilopoiesis, and that this is likely not mediated simply through the blocking of IL-5 production at the airway.     

Monoclonal rat anti-human lymphocyte antibody Campath-1 binds to T and B lymphocytes but effectively lyses only T cells

Binding and human complement-mediated T and B lymphocyte lysis were investigated in bone marrow samples obtained from 15 normal donors and 12 patients with a variety of malignant disorders undergoing marrow cryopreservation prior to autologous bone marrow transplantation. All marrow samples were obtained during remission except for one patient with neuroblastoma. The mononuclear cell fractions were collected and the distribution of B cell restricted markers (surface Ig and GP-70) and T cell surface markers (Leu-1 and rosettes with sheep red blood cells) were studied before and after marrow purging with Campath-1, a monoclonal rat anti-human lymphocyte antibody, and autologous serum as complement. Effective lysis of T lymphocytes (greater than 99.5%) was documented in all cases. However, although effective binding of Campath-1 to B lymphocytes was uniform, no effective lysis occurred. The data suggest that effective lysis of mature T lymphocytes can be accomplished while leaving normal B lymphocytes intact.