Risk factors of COVID-19 mortality: a systematic review of current literature and lessons from recent retracted articles.

OBJECTIVE: Recently, two influential articles that reported the association of (hydroxy)chloroquine or angiotensin converting enzyme (ACE) inhibitors and coronavirus disease 2019 (COVID-19) mortality were retracted due to significant methodological issues. Therefore, we aimed to analyze the same clinical issues through an improved research method and to find out the differences from the retracted papers. We systematically reviewed pre-existing literature, and compared the results with those of the retracted papers to gain a novel insight. MATERIALS AND METHODS: We extracted common risk factors identified in two retracted papers, and conducted relevant publication search until June 26, 2020 in PubMed. Then, we analyzed the risk factors for COVID-19 mortality and compared them to those of the retracted papers. RESULTS: Our systematic review demonstrated that most demographic and clinical risk factors for COVID-19 mortality were similar to those of the retracted papers. However, while the retracted paper indicated that both (hydroxy)chloroquine monotherapy and combination therapy with macrolide were associated with higher risk of mortality, our study showed that only combination therapy of hydroxychloroquine and macrolide was associated with higher risk of mortality (odds ratio 2.33; 95% confidence interval 1.63-3.34). In addition, our study demonstrated that use of ACE inhibitors or angiotensin receptor blockers (ARBs) was associated with reduced risk of mortality (0.77; 0.65-0.91). CONCLUSIONS: When analyzing the same clinical issues with the two retracted papers through a systematic review of randomized controlled trials and relevant cohort studies, we found out that (hydroxy)chloroquine monotherapy was not associated with higher risk of mortality, and that the use of ACE inhibitors or ARBs was associated with reduced risk of mortality in COVID-19 patients.

Aqueous Superparamagnetic Magnetite Dispersions with Ultrahigh Initial Magnetic Susceptibilities.

Superparamagnetic nanoparticles with a high initial magnetic susceptibility χo are of great interest in a wide variety of chemical, biomedical, electronic, and subsurface energy applications. In order to achieve the theoretically predicted increase in χo with the cube of the magnetic diameter, new synthetic techniques are needed to control the crystal structure, particularly for magnetite nanoparticles larger than 10 nm. Aqueous magnetite dispersions (Fe3O4) with a χo of 3.3 (dimensionless SI units) at 1.9 vol %, over 3- to 5-fold greater than those reported previously, were produced in a one-pot synthesis at 210 °C and ambient pressure via thermal decomposition of Fe(II) acetate in triethylene glycol (TEG). The rapid nucleation and focused growth with an unusually high precursor-to-solvent molar ratio of 1:12 led to primary particles with a volume average diameter of 16 nm and low polydispersity according to TEM. The morphology was a mixture of stoichiometric and substoichiometric magnetite according to X-ray diffraction (XRD) and Mössbauer spectroscopy. The increase in χo with the cube of magnetic diameter as well as a saturation magnetization approaching the theoretical limit may be attributed to the highly crystalline structure and very small nonmagnetic layer (∼1 nm) with disordered spin orientation on the surface.

USP8 suppresses death receptor-mediated apoptosis by enhancing FLIPL stability.

FLICE-like inhibitory protein (FLIP) is a critical regulator of death receptor-mediated apoptosis. Here, we found ubiquitin-specific peptidase 8 (USP8) to be a novel deubiquitylase of the long isoform of FLIP (FLIPL). USP8 directly deubiquitylates and stabilizes FLIPL, but not the short isoform. USP8 depletion induces FLIPL destabilization, promoting anti-Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and tumor necrosis factor alpha-induced extrinsic apoptosis by facilitating death-inducing signaling complex or TNFR1 complex II formation, which results in the activation of caspase-8 and caspase-3. USP8 mRNA levels are elevated in melanoma and cervical cancers, and the protein levels of USP8 and FLIPL are positively correlated in these cancer cell lines. Xenograft analyses using ME-180 cervical cancer cells showed that USP8 depletion attenuated tumor growth upon TRAIL injection. Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIPL and inhibits extrinsic apoptosis by stabilizing FLIPL.

USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics.

Given their crucial role in apoptosis suppression, inhibitor of apoptosis proteins (IAPs) have recently become attractive targets for cancer therapy. Here, we report that cellular IAP2 (cIAP2) is specifically stabilized in several cancer cell lines, leading to resistance to Smac mimetics, such as BV6 and birinapant. In particular, our results showed that cIAP2 depletion, but not cIAP1 depletion, sensitized cancer cells to Smac mimetic-induced apoptosis. Ubiquitin-specific protease 11 (USP11) is a deubiquitylase that directly stabilizes cIAP2. USP11 overexpression is frequently found in colorectal cancer and melanoma and is correlated with poor survival. In our study, cancer cell lines expressing high levels of USP11 exhibited strong resistance to Smac mimetic-induced cIAP2 degradation. Furthermore, USP11 downregulation sensitized these cells to apoptosis induced by TRAIL and BV6 and suppressed tumor growth in a xenograft model. Finally, the TNFα/JNK pathway induced USP11 expression and maintained cIAP2 stability, suggesting an alternative TNFα-dependent cell survival pathway. Collectively, our data suggest that USP11-stabilized cIAP2 may serve as a barrier against IAP-targeted clinical approaches.

Small cell carcinoma of the ureter with squamous cell and transitional cell carcinomatous components associated with ureteral stone.

We report a case of primary small cell carcinoma of the ureter with squamous cell and transitional cell carcinomatous components associated with ureteral stone, which is unique in that the patient has remained free of tumor recurrence for 36 months after the surgery without adjuvant chemotherapy or radiotherapy. A 60-yr-old man presented himself with a right flank pain. Computed tomography revealed an ill-defined mass and a stone in the lower one third of the right ureter, and hydronephroureterosis above the stone-impacted site. The patient underwent right nephroureterectomy and stone removal. Upon gross examination, a 3.8 x 1.8 x 1.2 cm white and partly yellow mass was noted in the anterior part of the ureter, resulting in indentation of the ureteral lumen on the posterior side. Light microscopic examination revealed that the mass was mainly composed of small cell carcinoma, and partly squamous cell and transitional cell carcinomatous components. The overlying ureteral mucosa and renal pelvis also contained multifocal dysplastic transitional epithelium and transitional cell carcinoma in situ. There was no vascular invasion, and the surgical margins were free of tumor. The small cell carcinomatous component was positive for chromogranin, neuron specific enolase, synaptophysin, and pancytokeratin but negative for high molecular-weight cytokeratin (K-903) by immunohistochemistry.

Prostate shape and symptom score in benign prostatic hyperplasia.

Prostates of the same volumes were found to have very variable shapes, that is, combinations of variably elongated width, height, and lengths. These were believed to be possible causes of the differences in the severity of both the obstructions and symptoms in the prostates even when their volumes were similar. We measured the transverse (width), anterior-posterior (height) and longitudinal (length) diameters of the prostates and the transition zone, and their calculated volumes using transrectal ultrasonography. To establish the relationship between the International Prostate Symptom Score (IPSS) and each of the dimensional parameters of the transition zone and the total prostate, 105 consecutive patients (mean age 66.43 +/- 9.24 years with a range o6f 46 to 90) who had voiding dysfunctions that were presumably related to BPH were analyzed using the t-test. Patients with conditions other than BPH were excluded. The results were as follows: 1. There was no significant correlation between the IPSS and any prostate volume parameter in the constant prostate volume conditions, because of the small numbers in each group. However, in the analysis of the total number of cases in all the volume categories, a significant correlation was found between the IPSS and some prostate dimensions; i.e., the longitudinal parameters in the total prostates (p < 0.01), and the transverse (p < 0.05) and longitudinal parameters (p < 0.05) in the transition zones. 2. Further investigations of the statistics of these significant parameters showed that prostates that were longer than 4 cm had significantly more severe symptoms than prostates shorter than 4 cm (p < 0.05), and that prostates with a ratio of length in the transition zone to the length in the total prostate ratio that was greater than 0.8 had significantly higher symptom scores than those with lower ratios (p < 0.05). When evaluating patients who have BPH, it is important to consider the shape of prostate. More aggressive treatment may be indicated in cases where the transition zone lengths exceeds 4 cm and the transition zone to total prostate length ratio exceeds 0.8.

Analysis of Philadelphia chromosome-negative BCR-ABL-positive chronic myelogenous leukemia by hypermetaphase fluorescence in situ hybridization.

BACKGROUND: In 5%-10% of patients with of chronic myelogenous leukemia (CML), the Philadelphia chromosome (Ph) is not identified, despite the presence of the associated BCR-ABL molecular abnormality (Ph-negative, BCR-ABL-positive CML) because of sub-microscopic rearrangements. PATIENTS AND METHODS: Six patients with Ph-negative, BCR-ABL-positive CML were investigated. The Ph chromosome detection via fluorescence in situ hybridization after 24-hour mitotic arrest of bone marrow cultures resulting in several hundreds of metaphases (hypermetaphase FISH or HMF) was useful in explaining the nature of the six cases. RESULTS: Four patients had a low frequency of Ph-positive cells by HMF (5.7%, 4.8%, 3.9%, 0.2%), i.e., a typical Ph translocation. However, two cases involved a 9q34 inserted into chromosome 22q11 (74.2% and 92%), without a deletion from chromosome 22 and reciprocal translocation onto 9, i.e., not a typical Ph translocation. The pattern of UBCR gene rearrangement was characterized by the same genomic recombination of 5-BCR and c-ABL, both in the four cases of typical translocation (9;22) and in the two cases of insertion of 9q34 into chromosome 22q11. CONCLUSIONS: The HMF identified two different bases for Ph-negative, BCR-ABL-positive cells in CML-presence of low frequency of cells with typical Ph translocations or presence of cells with ABL insertions into the BCR gene on chromosome 22.

Philadelphia chromosome-positive myeloid cells in the peripheral blood of chronic myelogenous leukemia patients: comparison with the frequency detected in cycling cells of the bone marrow.

Monitoring the frequency of the Philadelphia (Ph) chromosome in chronic myelogenous leukemia (CML) is important in determining the effectiveness of treatment for patients during therapy. This can be done with high resolution by subjecting short-term bone marrow cultures (48 h) to 24 h of mitotic arrest before harvest and detecting Ph-positive (Ph+) metaphases by fluorescence in situ hybridization (FISH) in a procedure termed hypermetaphase FISH or HMF. Here, we compared procedures for detecting Ph+ interphase cells (interphase FISH or I-FISH) in peripheral blood polymorphonucleocytes (PMNs) with HMF results on the bone marrow of the same 26 CML patients in different stages of remission. The probes for I-FISH in these experiments were relatively large (200-300 kb) and sufficiently resolved in PMNs so that 97.5% of the cells were scorable. The correlation between the frequencies of Ph+ cells from the two different cell sources was excellent (r = 0.983, P < 0.0001); however, there was a consistently higher level of Ph+ cells observed in the cycling marrow cells than in the peripheral blood PMNs. This was discussed in terms of current theories of apoptosis in CML cells. The large number of PMNs analyzable by I-FISH (approximately 500/patient in this study) provided sufficiently narrow 99% confidence intervals to suggest the procedure as an effective and efficient method for monitoring the frequency of Ph+ cells in CML patients undergoing therapy. However, for detection and quantification of minimal residual disease, HMF is preferable to I-FISH because of the much lower frequency of false-positive readings with the former procedure.

Role of nitric oxide in penile erection.

The present study was undertaken to investigate the role of nitric oxide (NO) in erectile physiology by correlating its action with the existence and activity of nitric oxide synthase (NOS), which produces NO. We applied Western blot analysis in both human and rat penile tissue. In the rat, reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining and spectrophotometric assay were also performed, in addition to in vivo electroerection study with pharmacological manipulation. Western blot analysis identified a protein of 155 KDa identical to the neural form of NOS in the human and rat penis. The NOS blot densities in the two species were similar, and both were lower than that in the rat cerebellum. Histochemical staining localized NOS to neurons innervating the corpora cavernosa, including the pelvic plexus, the cavernosal nerves and their terminal fibers within the corporeal erectile tissue, and dorsal penile nerves. NOS activity was also found in the cerebellum, urethra, penis, and urinary bladder, in decreasing order of intensity. Intracavernous injections of NOS inhibitor (L-NOARG or L-NAME in concentrations from 10(-6) M to 10(-3) M suppressed electrostimulation-induced erection in a concentration-dependent manner. Subsequent intracavernous injection of L-Arginine (10(-2) M) partially restored the erection. The neural form of constitutive NOS in the corpora cavernosa synthesizes NO, which mediates penile erection. Determination of cavernosal NOS expression or activity may permit characterization of certain pathological conditions that cause impotence.

Allogeneic transplantation for recurrent or refractory non-Hodgkin's lymphoma with poor prognostic features after conditioning with thiotepa, busulfan, and cyclophosphamide: experience in 44 consecutive patients.

We report the outcomes of 44 consecutive patients with non-Hodgkin's lymphoma (NHL) who participated in prospective studies of allogeneic transplantation after conditioning with thiotepa, busulfan and cyclophosphamide. Within a range of 27-57 years, the median age was 37. Of the 44 patients, 12 (27.2%) had high-grade lymphomas, 27 (61.4%) had intermediate-grade lymphomas, and five (11.3%) had low-grade lymphomas. Twenty-eight (63.6%) patients had chemotherapy refractory disease. Thirty (68.2%) patients had stage IV disease at the time of transplantation, involving the bone marrow in 19 (43.2%). Eight (18.1%) patients had undergone previous transplantation, and 13 (29.5%) patients had received high-dose CVP as induction within 2 months prior to transplantation. Thirty-eight (86.3%) patients had an HLA-identical donor, and 6 (13.6%) had a one-antigen mismatched related donor. Twenty (45.4%) patients received bone marrow and 24 (54.6%) received granulocyte colony-stimulating factor (G-CSF) mobilized stem cells. The graft-versus-host disease (GVHD) prophylaxis contained cyclosporine or tacrolimus in combination with either methylprednisolone in 32 (72.7%) patients or with methotrexate in 12 (27.2%) patients. The actuarial probability of disease-free survival at 2 years is 23% (95% CI 13%-40%). Donor stem cell use was associated with a significantly decreased risk of treatment-related toxicity (p < 0.001), but with an increased risk for GVHD and delayed fungal and viral infections. These infections are linked not only to the use of donor-stem cells, but also to the methylprednisolone in the GVHD prophylaxis regimen. Improvements in the outcome of patients with advanced NHL and undergoing allogeneic transplantation will depend on the development of effective and non-toxic regimens for conditioning, GVHD prophylaxis, and opportunistic infections prophylaxis.

Emergence of new clonal abnormalities following interferon-alpha induced complete cytogenetic response in patients with chronic myeloid leukemia: report of three cases.

New treatments which may change the course of a disease, or which have potential carcinogenicity, may result in the development of new cytogenetic or clinical disorders. Three patients with Philadelphia chromosome-positive (Ph-positive) chronic myeloid leukemia (CML) who developed new cytogenetic abnormalities after achieving a cytogenetic complete remission (CR) of their Ph-positive disease with interferon alpha (IFN-alpha) based therapy are described. Patient 1 developed chromosomal abnormalities involving chromosomes 5 (5q13-34) and later 7 (monosomy 7) 60 months after the start of therapy and 20 months after IFN-alpha was discontinued. A myelodysplastic syndrome was noted 83 months from the start of therapy. Patient 2 developed a myeloproliferative syndrome with 18p11 chromosomal abnormalities 90 months after the start of the therapy and 60 months after IFN-alpha was discontinued. Patient 3 developed a chromosome 11 abnormality (11q21-23) 23 months after the start of therapy, without hematological manifestations. All three patients remain in cytogenetic CR of Ph-positive disease with the hypermetaphase fluorescent in situ hybridization and polymerase chain reaction studies for BCR/ABL showing minimal residual disease. The emergence of new cytogenetic or clinical disorders in patients with CML on IFN-alpha therapy needs to be monitored. These findings may be related to changing the natural course of CML, to therapy, or to the emergence of suppressed clones in a stem cell disorder.

Collection of peripheral blood stem cells from normal donors 60 years of age or older.

We report 14 normal peripheral blood stem cell (PBSC) donors > or = 60 years of age who had cytokine mobilization followed by PBSC apheresis for allogeneic transplantation. Mobilization was achieved with filgrastim (6 microg/kg twice daily). Their median age was 63.5 years (range 60-77), and 43% had a positive medical history, mainly hypertension and/or cardiac problems. Their median pre-apheresis leucocyte count (x 10(9)/l) was 38.6 (range 29.6-63.4). The median apheresis yield (x 10(6) CD34+ cells/litre blood processed, first apheresis) was 27.9 (range 1.6-54.8). The target cell dose (> or = 4 x 10(6) CD34+ cells/kg recipient) was reached with one procedure in eight (57%) donors. Filgrastim-related adverse events were acceptable and apheresis was well tolerated. When compared to younger donors (< 60 years of age), a trend to a lower CD34+ apheresis yield and a requirement for more than one apheresis to achieve the collection target (> or = 4 x 10(6) CD34+ cells/kg) was evident. Although older (> or = 60 years) donors seem to mobilize less effectively, these data suggest that PBSC collection from them is feasible and has an acceptable short-term safety profile.

A dose-response study of alprostadil sterile powder (S.Po.) (Caverject) for the treatment of erectile dysfunction in Korean and Indonesian men.

This open-label, dose-escalation study investigated the efficacy and safety of alprostadil (PGE1, prostaglandin E1) Sterile Powder (S.Po.) (Caverject) for treatment of erectile dysfunction (ED) in 84 men with ED of various etiologies lasting > or = 4 months. Doses started with 2.5 micrograms, then 5 micrograms, 10, 15, 20, 30, up to a 40 micrograms maximum. Eligible patients received single alprostadil injections in the physician's office until an erection occurred. Ten minutes after injection, the patient's erection was clinically evaluated. Optimal response was defined as erection sufficient to permit vaginal penetration and lasting 30-60 min. The patient also reported his own evaluation of response and any side effects. Patients were 24-65 y old (mean: 43.7 y), had ED of psychogenic, vascular, or neurogenic origin lasting 4 months-30 y (mean: 3.75 y). Of 84 patients enrolled, 82 completed the study. In the 82 patients who completed the study 78 (92.9%) achieved an optimal response; 18/78 patients (23.1%) had an optimal response at 2.5 micrograms, 9/78 (11.5%) at 5 micrograms, 21/78 (26.9%) at 10 micrograms, 12/ 78 (15.4%) at 15 micrograms, and 11/78 (14.1%) at 20 micrograms. Only 5/78 (6.4%) at 30 micrograms and 2/78 (2.6%) at 40 micrograms achieved an optimal response. Mean optimal alprostadil dose was 11.9 micrograms, and the mean minimal effective dose was 9.9 micrograms. Mean onset of erection was 11.2 min; mean duration of erection was 50.5 min. Penile pain in five patients (6%) was the only reported side effect.

Usefulness of detection of minimal residual disease by 'hypermetaphase' fluorescent in situ hybridization after allogeneic BMT for chronic myelogenous leukemia.

Hypermetaphase FISH (HMF), a molecular cytogenetic procedure combining the long term mitotic arrest of bone marrow cultures with detection of a specific chromosomal rearrangement by fluorescence in situ hybridization (FISH), has recently been shown to be effective in determining the frequency of Philadelphia chromosome positive (Ph+) cells in the bone marrow of chronic myelogenous leukemia (CML) patients undergoing treatment. By combining the probe for the Ph chromosome with one for the detection of the X chromosome, we used HMF to monitor the presence of malignant cells within the emerging host cell population in the marrow of a CML patient that had undergone sex-mismatched allogeneic bone marrow transplantation. In successive studies, we detected 0.5% and 1.75% Ph+ cells, respectively, confirmed by Western blot analysis for p210 protein. These readings occurred concordantly with a repopulation of host-derived diploid female cells. Standard G-band cytogenetic analyses did not detect any Ph+ cells at these time points. Intervention with donor lymphocyte infusion reinduced complete remission. This experience indicates that HMF is useful to identify low levels of repopulation by Ph+ cells in the marrow post-BMT at a stage when intervention is most efficacious.

Phase I trial of cyclosporine-induced autologous graft-versus-host disease in patients with multiple myeloma undergoing high-dose chemotherapy with autologous stem-cell rescue.

PURPOSE: To determine the feasibility and toxicity of inducing autologous graft-versus-host disease (GVHD) with cyclosporine in patients with multiple myeloma undergoing autologous stem-cell transplantation. PATIENTS AND METHODS: Fourteen multiple myeloma patients with a median age of 50 years (range, 41 to 63) were enrolled. The median time from diagnosis to transplant was 651 days (range, 229 to 3,353). Ten patients had primary refractory disease, two were in first remission, and two were responsive to salvage therapy. The preparative regimen consisted of thiotepa, busulfan, and cyclophosphamide. Cyclosporine was administered daily for 28 days after the stem-cell infusion, and the dose was adjusted to maintain whole-blood cyclosporine levels between 50 and 150 ng/dL in the first seven patients (low-level group) and between 150 and 300 ng/dL in the other seven patients (high-level group). RESULTS: All patients achieved neutrophil engraftment a median of 11 days after transplant. Four patients developed > or = grade 2 hepatic toxicity, six developed > or = grade 2 nephrotoxicity, and four developed reversible cardiac toxicity. Only one treatment-related death occurred. Cyclosporine was withheld in seven patients for a median of 6 days because of renal and/or liver dysfunction. One patient developed clinical skin GVHD, which responded to corticosteroid therapy. Six patients developed histologic evidence of GVHD without clinical signs of GVHD (subclinical GVHD). The incidence of clinical and subclinical GVHD was similar in both cyclosporine groups. Three of 11 patients assessable for response achieved remissions. Three patients experienced disease progression 80, 160, and 354 days after transplant. Ten patients are alive without progression between 56 and 444 days after transplant. CONCLUSION: Induction of autologous GVHD by posttransplant cyclosporine is feasible and well tolerated in patients with multiple myeloma.

Peripheral blood stem cell apheresis in normal donors: feasibility and yield of second collections.

We report 13 normal peripheral blood stem cell (PBSC) donors who had a second PBSC collection for allogeneic transplantation performed after the first. The median interval between the first and second collection was 5 months. Mobilization was achieved with filgrastim (12 microg/kg/d). No significant difference was found in the median pre-apheresis leucocyte count (x10(9)/l) between the two donations (40.2 v 38.5; P = 0.91). The median apheresis yield (x10(6) CD34+ cells/litre blood processed, first apheresis) was also similar (28 v 27.3; P = 0.91). Filgrastim-related adverse events were comparable. These data suggest that second PBSC collections are feasible, similarly tolerated and provide comparable apheresis yields.

Delayed effects of rhG-CSF mobilization treatment and apheresis on circulating CD34+ and CD34+ Thy-1dim CD38- progenitor cells, and lymphoid subsets in normal stem cell donors for allogeneic transplantation.

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) is emerging as a new stem cell transplant modality. Rather than undergoing general anesthesia for bone marrow harvest, normal blood stem cell donors are subjected to rhG-CSF mobilization treatment followed by single or multiple apheresis. Whereas the effects of cytokine treatment and apheresis on stem cell peripheralization and collection have been described, little is known about delayed effects of rhG-CSF treatment and apheresis on a normal hematopoietic system, and there are no long-term data that address safety issues. Ten normal, patient-related donors underwent a 3 or 4 day rhG-CSF (filgrastim) treatment (12 micrograms/kg/day) followed by single or tandem apheresis. We monitored peripheral blood (PB) cellularity including CD34+ and lymphoid subsets at baseline, during cytokine treatment, prior to apheresis, and at days 2, 4, 7, 30 and 100 post-apheresis. The PB progenitor cell concentration peak prior to apheresis was followed by a nadir by day 7 and normalized by day 30, with the exception of the most primitive CD34+ Thy-1dim CD38- progenitor subset that reached a nadir by day 30. Lymphoid subsets such as CD3, 4, 8, suppressor cells (CD3+ 4- 8- TCR+ alpha beta), and B cells (CD19+) showed a similar pattern with a nadir concentration by day 7, followed, except for B cells, by a rebound by day 30 and subnormal counts at day 100. The PB concentrations of hemoglobin and platelets dropped mainly due to the apheresis procedure itself, and normalized by day 30. With cytokine treatment, the PB alkaline phosphatase and lactate dehydrogenase concentrations increased 2.2- and 2.8-fold, respectively, over baseline, and returned to normal range by day 30. Based on the preliminary nature of this study, the clinical relevance of these findings is still unclear.

Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy.

To formally test the hypothesis that the granulocyte/macrophage colony-forming unit (GM-CFU) cells can contribute to early hematopoietic reconstitution immediately after transplant, the frequency of genetically modified GM-CFU after retroviral vector transduction was measured by a quantitative in situ polymerase chain reaction (PCR), which is specific for the multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU methylcellulose plating assay. The results of this analysis showed no difference between the transduction frequency in the products of two different transduction protocols: "suspension transduction" and "stromal growth factor transduction." However, when an analysis of the frequency of cells positive for the retroviral MDR-1 vector posttransplantation was carried out, 0 of 10 patients transplanted with cells transduced by the suspension method were positive for the vector MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the cells transduced by the stromal growth factor method were positive for the MDR-1 vector transcription unit by in situ or in solution PCR assay (a difference that is significant at the P = 0.0065 level by the Fisher exact test). These data suggest that only very small subsets of the GM-CFU fraction of myeloid cells, if any, contribute to the repopulation of the hematopoietic tissues that occurs following intensive systemic therapy and transplantation of autologous hematopoietic cells.

Allogeneic peripheral blood stem cell transplantation using normal patient-related pediatric donors.

Successful allogeneic peripheral blood progenitor cell (PBPC) transplantation has recently been reported by several transplant centers. This is a first report describing allogeneic PBPC transplantation in five patients using related pediatric donors between the ages of 4 and 13 years. Donors underwent 3 or 4 days of rhG-CSF treatment (6 micrograms/kg q 12 h) for stem cell peripheralization prior to PBPC collection, which was performed by continuous-flow apheresis on day 4 or 5. Venous access was exclusively by ante-cubital veins. A median of 2.2 times (range 1.4-3.6) the donor's total blood volume (TBV) was processed per procedure. In cases where the donor's TBV was < 2 liters, the blood cell separator was primed with human serum albumin (HSA-5%), and anticoagulation was performed using a combination of heparin (pre-apheresis bolus + continuous infusion (CI)) and/or ACD-A (CI at a reduced rate). The median number of CD34+ cells collected per kg of donor body weight (b.w.) and per liter of donor blood processed during each procedure was 128 x 10(4) (range 58 x 10(4)-314 x 10(4)). Between one and two aphereses were sufficient to collect a safe CD34+ cell engraftment dose of 3 or 4 x 10(6)/kg of recipient b.w. Two PBPC recipients were parents, and three were siblings. After freezing and thawing, the median number of CD34+ cells per kg of recipient b.w. thawed and transfused was 8.5 x 10(6) (range 3.2 x 10(6)-9.7 x 10(6)). The time to PMN > 1000/microliters was between 10 and 16 days (four out of five evaluable patients), and platelets > 20000/microliters were reached between day 13 and 14 post-transplantation (three out of five evaluable patients). Two out of three evaluable patients developed grades one and three acute GVHD, and one out of three developed chronic GVHD. Two patients died of sepsis and VOD at day 10 and 19, respectively. Two adult patients are alive and in cytogenetic and molecular remission of CML at +339 and +227 days post-allotransplantation. One 3-year-old girl with hemophagocytic lymphohistiocytosis is in remission at +304 days post-transplantation. Using pediatric donors for allogeneic PBPC transplantation appears to be safe, yields a sufficient amount of progenitors for prompt engraftment, and results in clinical outcome similar to adult PBPC allotransplantation.