Search

Forty-one patients were studied at set times after allogeneic blood stem cell transplantation (alloBSCT) for recovery of lymphocyte numbers and function. Cells were mobilized with G-CSF from HLA-matched related donors and cryopreserved. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate; G-CSF was administered post-transplant. Median time to absolute lymphocyte count (ALC) 500/μl was 17 days vs 41 and 49 days in historical alloBMT patients with G-CSF (n = 23) or no cytokine (n = 29) post-transplant, respectively (P < 0.0001). CD4/CD8+ ratio was 1.9 on day 28 after alloBSCT, then gradually declined to 0.8 at 1 year due to more rapid CD8+ cell recovery. Mean phytohemagglutinin-induced T cell responses were lower than normal on day +28 (P < 0.05), then tended to recover towards normal values. Natural-killer cytotoxicity remained low from day +28 to 1 year post-alloBSCT, but considerable lymphokine-activated killer cytotoxicity was induced from cells already obtained on day +28. Faster lymphocyte recovery correlated with better survival in alloBSCT patients (median follow-up 287 days, P = 0.002), ALC recovery was not affected by acute GVHD, CMV infections or doses of infused cells. ALC recovery did not correlate with survival in either historical alloBMT group. These data suggest that after alloBSCT lymphocyte reconstitution is faster than after alloBMT, and that quicker lymphocyte recovery predicts better survival in the alloBSCT setting. [ABSTRACT FROM AUTHOR]

Background: Circulating uric acid (UA) is an important biomarker, not only in the detection and management of gout, but also in assessing the risk of related comorbidity. The impact of collection methods on clinical UA measurements has been the subject of limited study. After observing significant differences between UA concentrations of blood samples obtained by different collection tubes, we began examining the effects of exogenous tube components on measured UA concentrations. We aimed to: (1) demonstrate the variability in uricase-based UA measurements attributable to different collection methods and (2) identify factors influencing this variability., Methods: Blood samples from human subjects were collected using Serum Separator Tubes (SST tubes), Acid Citrate Dextrose (ACD) tubes, and Sodium Citrate (SC) tubes. Circulating UA concentrations were measured by chemistry analyzers utilizing the uricase method. Absorbance assays were run in order to determine the effects of citric acid, sodium citrate, and dextrose on measured absorbance in the presence of leuco crystal violet dye, hydrogen peroxide, and peroxidase. Statistical analyses-including Student's T tests and ANOVA-were used to compare results., Results: UA concentrations of blood samples collected in ACD tubes were significantly lower than those collected in SST tubes (P < 0.01). Samples collected in SC tubes trended towards lower UA measurements than samples collected in SST tubes, although this difference did not reach statistical significance (P = 0.06). Blood samples spiked with separate concentrations of sodium citrate (3.2 and 22.0 g/L), citric acid (8.0 g/L), and dextrose (24.5 g/L) demonstrated significantly lower UA measurements compared to controls (P < 0.01). Absorbance assays demonstrated that increasing concentrations of citric acid and sodium citrate-in the presence of leuco crystal violet, hydrogen peroxide, and peroxidase-decreased the amount of oxidized dye in the uricase method of UA measurement in a dose-dependent manner (P < 0.01). In contrast, dextrose did not significantly alter the amount of oxidized dye available., Discussion: Our results indicate that citric acid obstructs accurate uricase-based UA measurement, providing falsely low values. Citric acid, a known antioxidant, scavenges hydrogen peroxide, a key intermediate using the uricase method. By scavenging hydrogen peroxide, citric acid decreases the amount of oxidized leuco dye leading to falsely low UA measurements. Therefore, collection tubes, like ACD and SC tubes, which contain concentrations of citric acid or its conjugate base sodium citrate should not be used to measure circulating UA levels when utilizing uricase-based measurement methods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: The purpose of this study was to determine whether immunophenotypic profiles detected by flow cytometry are useful in differentiating chronic myelomonocytic leukemia (CMML) from reactive monocytosis, and between CMML subtypes., Methods: Eight-color flow cytometry was used to immunophenotype blasts, monocytes, and granulocytes in the bone marrow of 34 patients with CMML and 12 patients with reactive monocytosis., Results: Bone marrow myeloblast, promonocyte, and monocyte counts by flow cytometry were significantly higher in the CMML group than in the reactive monocytosis group. Myeloblast aberrancies were present in all CMML patients as compared with 2 of 12 (16.7%) reactive monocytosis patients (P < 0.001). The number of blast aberrancies ranged from one to nine (median, four) in CMML patients and 94.1% of CMML cases exhibited ≥ two aberrancies. In contrast, two reactive monocytosis cases showed only one phenotypic abnormality of blasts. Monocyte and granulocyte aberrancies were present in 26 of 34 (76.5%) and in 31 of 34 (91.2%) CMML patients, respectively. Decreased side scatter (SSC) and abnormal CD11b/CD13/CD16 maturation pattern in granulocytes were more frequent in CMML than in reactive monocytosis. No significant differences in antigen expression were detected between the CMML subtypes except that altered CD45/SSC pattern on the blasts was more commonly observed in CMML-0/1 than in CMML-2., Conclusions: CMML has phenotypic aberrancies in monocytes, granulocytes, and more frequently in myeloblasts. Aberrant expression of two or more antigens in myeloblasts by flow cytometry has a high sensitivity (94.1%) and a high specificity (100%) to differentiate CMML from reactive monocytosis. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Dendritic cells (DCs) are critical to the initiation of a T-cell response. They constitute the most potent antigen-presenting cell (APC) endowed with the unique capacity to stimulate an antigen-specific T-cell responses by naïve T cells. Adenoviruses (Ad) have high transduction efficiency for many cell types including cells of hematopoietic origin independent of their mitotic status, and replication-defective Ad have demonstrated a safety profile clinically. Further, Ad vectors provide a high level of transgene expression, and Ad-transduced DCs can effectively present antigenic proteins. In this chapter, we outline a functionally closed, good manufacturing protocol for the differentiation of monocytes into DCs and transduction by Ad vectors. Basic functional and phenotypic release assays are provided, as well as contrasting research approaches for Ad-transduced DC-based vaccines.

Anticancer immunotherapy using dendritic cell (DC) based vaccines provides an adjuvant therapeutic strategy that is not cross reactive with conventional therapeutics. However, manufacturing of DC vaccines requires stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization. Optimally this includes a closed system for monocyte isolation, in combination with closed culture and washing systems and an effective vector transduction strategy. In this study, we used the Gambro Elutra to enrich monocytes from non-mobilized leukapheresis products collected from healthy donors. This approach enriched monocytes from an average frequency of 13.6+3.2% (mean+SEM), to an average frequency of 79.5+4.3% following enrichment with a yield of 79 to 100%. The monocytes were then cultured in a closed system using gas permeable Vuelife fluoroethylene propylene (FEP) bags and X-vivo-15 media containing 10 ng/ml granulocyte-macrophage colony-stimulation factor (GM-CSF) and 5 ng/ml Interleukin (IL) 4. The cultures were re-fed on days two and four, with a 25% media volume and cytokines. Following culture for seven days, the cells were harvested using a Cobe-2991 and concentrated using a bench centrifuge retrofitted with blocks to allow centrifugation of 72 ml bags and supernatant removed using a plasma extractor. This approach reduced the media volume to an average of 17.4 ml and an average DC concentration of 6.3+1.0x10(7) cells/ml, a viability of 93.8+2.2%, a purity of 88.9+3.3% and a total yield of 8.5+1.4x10(8) DCs. Based on the identification of DR+ cells as DCs we had an average yield of 46+8% using a calculation based on the number of monocytes in the apheresis product and the resulting DCs differentiated from monocytes. The use of DCs as a vaccine, required transduction with an adenovirus (Adv) vector with the tumor suppressor, p53 transgene (Adv5CMV-p53) as the antigen at a DC concentration of 9x10(6) DCs/ml at an Ad5CMV-p53: DC ratio of 20,000:1, and a 2 or 3 hour co-culture, followed by a 1:10 dilution with media and an additional 16-22 hour incubation. Following incubation, the DCs were washed twice and the supernatants removed using a plasma extractor. The average viability after infection with Ad5CMV-p53 was 87.9+/-2.6% with an average of 20.3+5.4% of the DCs expressing p53. The calculated yield of DCs following Ad5CMV-p53 transduction, based on the number of monocytes in the apheresis products, averaged 12.4+3.8%. We conclude that it is possible to efficiently manufacture Adv transduced DCs using a functionally closed system.

In this study, mononuclear cells (MNCs) from granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cell (BSC) harvests from 104 healthy donors were analyzed for their immunological functions and compared with MNCs from 28 steady-state nonmobilized donors. The relationships between donor characteristics (age, gender, weight, and HLA type) and immune functions of the harvests were also analyzed. There was a significant (P <.01) decrease in natural killer and lymphokine-activated killer (LAK) cell-mediated cytotoxicity for G-CSF-mobilized effector cells compared with nonmobilized cells. Similarly, there was a significant (P <.005) decrease in both T-cell and B-cell mitogen response in G-CSF-mobilized cells compared with nonmobilized cells. There was dose-dependent inhibition of LAK cell-mediated cytotoxicity, but this effect was not seen with other immune function assays. Changes in immune function did not appear to be determined by frequency of cellular phenotypes or expression of effector function genes seen in a reverse-transcription polymerase chain reaction. There was a significant relationship between expression of certain HLA alleles (A1, A3, A24, B44, B62, DR15, DR17; all P <.01) and increased immune function, such as cytotoxicity and/or mitogen response. A decrease in immune function with the HLA-DR13 expression was also observed (P <.01). Since the G-CSF increases the number of MNCs, the increase in effector cells might compensate for decreased immune functions of these cells in vivo when transplanted into patients. These results suggest a decreased immune function in G-CSF-mobilized BSC harvests and warrant further studies to correlate these data with clinical outcome.

Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.

Alveolar macrophages recovered by bronchoalveolar lavage from 14 heavy smokers with chronic bronchitis were assessed. Ultrastructural examination revealed marked cellular heterogeneity. Three subpopulations of alveolar macrophages were readily identifiable. These have been termed "young," "mature," and "degrading," reflecting their ultrastructural features. In addition, a majority of the cells were found to be positive by TUNEL staining, indicating DNA damage, but a very small percentage tested positive for Caspase-3, suggesting that apoptosis might not account for the DNA damage in at least some of these cells. A small percentage of proliferating cells were noted.

The group B coxsackieviruses (CVB) induce experimental pancreatitis and myocarditis in mice and are established agents of human myocarditis, especially in children. We tested the hypothesis that the development of CVB-induced myocarditis is linked to CVB-induced pancreatitis by studying the replication of different CVB strains in mice. Eight of nine genotypically different type 3 CVB (CVB3) strains induced acute pancreatitis in mice; of these, three viruses also induced acute myocarditis. One CVB3 strain was avirulent for both organs. Myocarditis was not observed in the absence of pancreatitis. The results obtained by inoculation of mice with strains of other CVB serotypes were consistent with these data. Infectious virus titers were measured in serum, pancreas, and heart as a function of time after inoculation of mice with three CVB3 strains. Each strain was representative of one of the three viral virulence phenotypes: avirulent, pancreovirulent only, and cardiovirulent. All strains replicated well and persisted in the pancreas through 8 days post-inoculation, but the cardiovirulent CVB3 strain tended to replicate to higher titer earlier and persist longer in sera, pancreatic, and cardiac tissues than the noncardiovirulent strains. Replication of the CVB3 strains were studied in two human pancreatic tumor lines and in primary human endothelial cell cultures derived from cardiac artery. Cardiovirulent strains, both individually and as a group, tended to replicate to titers as high as, or higher than, noncardiovirulent strains did in cell culture. The data are consistent with the possibility of an etiologic link between CVB-induced pancreatic and heart disease., (Copyright 2000 Wiley-Liss, Inc.)

A short phosphorothioate oligodeoxynucleotide telomere mimic with the sequence 5'-d(TTAGGG)-3', TAG-6, has been shown to inhibit telomerase activity and have antineoplastic and hematopoietic stimulatory properties. In this study, three immature male domestic swine (weighing approximately 40 kg) were administered 200 mg/m2 of TAG-6 by continuous intravascular infusion at rates of 0.48 +/- 0.07 mg/hr for 14 days to evaluate the pharmacokinetics, toxicity, and tissue distribution. There was considerable variability (both within each animal and across animals) observed in the pharmacokinetic data. The plasma half-life (t1/2 appeared to be short enough that it could be assumed that steady state was attained by at least 96 h after the start of the infusion. The t1/2 estimates for the three pigs were 8.96, 109, and 1.97 h (the long t1/2 for pig 2 may be explained by poor parameter estimation due to the variability). The volume of distribution ranged from 9.80 to 51.8 L (0.3-1.4 L/kg), and plasma clearance estimates ranged from 0.33 to 3.46 L/h (5.5-57.7 ml/min). The average plasma concentrations at steady state were 0.845, 0.933, and 0.178 microg/ml (0.44, 0.49, and 0.093 microM) for the three animals. Nearly 30% of the administered dose was cleared through renal excretion by day 7 postinfusion. The distribution of TAG-6 was primarily to the liver and kidney, but the spleen and thyroid accumulated relatively high concentrations of TAG-6. TAG-6 was metabolized to apparently higher molecular weight products, which were observed in the urine. The size periodicity of these apparently higher molecular weight products was in 6-base intervals, which is consistent with the actions of telomerase. The infusion did not produce significant changes in serum chemistry or circulating blood cells, but a decrease in colony-forming unit-granulocyte-monocyte (CFU-GM) colony formation from BM was observed. These data suggest that TAG-6 may be a very specific pharmacophore.

The study of alveolar macrophages (AM) from smokers by flow cytometry (FCM) has been limited by strong autofluorescence and the lack of reliable markers to identify macrophages. Crystal violet quenching was reported to be effective in reducing autofluorescence of AM. CD68 is a marker for macrophages in immunohistochemistry, but has been less useful in FCM because of poor surface expression. This study evaluated the effectiveness of a method for two-colour FCM analysis of AM combined with membrane permeabilization and crystal violet quenching. Bronchoalveolar lavage cells, fixed in 4% paraformaldehyde and permeabilized using 0.5% Triton X100, were incubated with fluorescent-labelled antibodies for 30 min and quenched with a saturated crystal violet solution. Two-colour FCM was then performed using forward/side scatter gating to select AM. Autofluorescence at 525 nm (fluorescein isothiocyanate) and 575 nm (phycoerythrin) markedly decreased after quenching. After permeabilization, 97.1+/-2.8% of the gated cells were CD68+, while 53.9+/-18.6% of the AM were positive without permeabilization. CD68+ cells were sorted and proved to be AM morphologically. Analysis of CD71 (transferrin receptor) expression by FCM correlated with immunocytochemistry (r=0.77, p<0.05). The permeabilization/quenching technique, therefore, represents a satisfactory means to evaluate alveolar macrophages by flow cytometry.

Background: CD34(+) cell enumeration in PBSC apheresis products has become the standard for assessing graft hematopoietic potential., Methods: An in-house, three color, lineage negative-gating technique [University of Nebraska Medical Center (UNMC) protocol] for CD34 cell enumeration was compared with the ISHAGE protocol over 100 apheresis products. Cell doses determined by both methods were compared with each other and to colony-forming units-granulocyte/macrophage (CFU-GM) assay results., Results: Overall, the assays compared well with each other for samples with CD34 cell doses > 0.2 2 10(6)/kg (r values > 0.8). The ISHAGE method showed a constant negative bias, with a mean of 38% in comparison to the UNMC protocol, which was more linear at lower cell doses. Both assays showed similar correlation with CFU-GM doses after log conversion (UNMC, r = 0.915; ISHAGE, r = 0.917). When comparing integer values, however, the ISHAGE method correlated with CFU-GM only in the high dose range (CFU-GM > 2 2 10(4)/kg), while the UNMC method correlated across the entire measured range of CFU-GM doses. Finally, an inter-technologist gating reproducibility study (n = 6) yielded a 23% coefficient of variation (CV) for the ISHAGE method and a 7% CV for the UNMC method, when the same two sets of CD34 histograms were analyzed to calculate cell dose., Discussion: In this study the lineage negative protocol (UNMC) had a larger dynamic range, correlated better with CFU-GM results and showed better inter-technologist reproducibility than the ISHAGE method.

Although the hematopoietic reconstituting ability of human umbilical cord blood cells (UCBC) is well documented, their antitumor cytotoxic potential has not been well studied. Therefore, UCBC were compared to normal peripheral blood stem cells (PBSC) and bone marrow (BM) stem cell harvests for cytomorphology, antitumor cytotoxic activity before and after ex vivo cytokine manipulation, response to T and B cell mitogens, expression of adhesion molecules and immunophenotypes using flow cytometry, cytokine production and in vivo antitumor activity. BM and PBSC, but not UCBC, did not form cellular clusters in culture. More cytotoxic granules were present in the cytoplasm of UCBC than PBSC following activation in vitro. Ex vivo manipulation of UCBC with cytokines produced more cytotoxicity to K562 and Raji tumor cells than PBSC or BM (p<0.001). Most cytotoxic cells in UCBC cultures were T lymphocytes, and a correlation existed between the number of CD56+ cells and cytotoxicity levels, particularly after in vitro activation with interleukin-2. No significant difference in adhesion molecule expression was noted among UCBC, PBSC and BM cells. However, there was a significantly decreased expression of CD54 molecules (ICAM) on UCBC compared to PBSC (p<0.05). IL-2 activated UCBC showed significant antitumor effects against K562 leukemic cells grown in SCID mice. Thus UCBC contained more antitumor effector cells and precursors than cells from marrow or peripheral blood cells which might be capable of providing a therapeutic effect.

Detection of small numbers of breast cancer cells in patient blood, aphereses, and bone marrow has become increasingly important as data have accumulated showing immunocytochemically (ICC) positive tumor cells in up to 50% of women with stage I and II breast cancer, who were initially thought to be cured of their disease but later relapsed. The ability to rule out the presence of micrometastatic disease at any stage of the clinical management protocol, whether before, during, or after therapy, would provide a useful monitoring and diagnostic tool for both the clinician and the scientist. Monitoring for the presence of minimal residual disease (MRD) is traditionally performed using ICC. A more recently established RT-PCR technique uses a molecular marker (the presence of the cytokeratin 19, CK19, transcript) to identify MRD in patient samples, with a level of sensitivity reported to be one tumor cell in 10(6) nucleated cells. This level of sensitivity is generally higher than that claimed for ICC. Based on the discriminating results of this first study, a number of laboratories have evaluated this technique for its diagnostic potential. Results from several laboratories showed a higher than expected false positive rate due to a variety of identified and unidentified sources. Therefore, the current study was designed to achieve two aims: to establish the level of sensitivity and specificity of the RT-PCR technique and to dissect out the possible variables that may contribute to a false positive result using this molecular approach. To accomplish the first goal, two simulation strategies were used, limited dilution of tumor cells into apheresis harvests and semi-quantitative PCR using stepwise dilutions of extracted RNA from tumor cells in apheresis harvests. The second goal was accomplished by performing sequential blood drawings with variably timed sample processing to identify some of the more common variables (time, anticoagulant, sample sequence) that may contribute to false positive results. Of three variables investigated, including type of blood preservative, sequence of blood tube collection, and time point of sample processing, each may contribute to a false positive result. In addition to these problems, known events involving illegitimate transcription of specific genes nonspecifically in tissue is also a potential source of false positive results. These issues may be further compounded by lack of attention to the more common methodologic problems encountered in any laboratory using the PCR technique. However, recommendations can be developed for the effective application of this technique, whose greatest strength is the demonstration of tumor negativity of the sample.

A phosphorothioate oligonucleotide (PS-ODN) with sequence identical to the repeat sequence of the mammalian telomere, 5'-d(TTAGGG)-3', was incubated with a Burkitt's lymphoma-derived (OMA-BL1) cell line. This hexanucleotide inhibits telomerase activity in cell lysates, lengthens cell doubling time, and induces apoptosis. Concatenated repeats (12-, 18-, and 24-mers) of the 5'-d(TTAGGG)-3' motif induce similar cellular responses. Scrambled sequences do not efficiently inhibit telomerase activity or significantly alter cell growth and viability. The in vivo efficacy of this PS-ODN was evaluated in a xenograft human-nude mouse model. Once tumors were established these animals were administered the telomere mimic, 5'-d(TTAGGG)-3', a control scrambled sequence 5'-d(TGTGAG)-3', or saline for 14 days via a subcutaneous osmotic pumps in a blinded study monitoring tumor size with dose and time. A significant decrease in tumor size was observed in animals given 50 micrograms/mouse/day 5'-d(TTAGGG)-3', but not following 5'-d(TGTGAG)-3', relative to the saline-treated animals. The antitumor activity of the 6-mer telomere mimic demonstrated a dose dependency including a reduction in metastatic nodules in the spleen. No activity was observed with the scrambled controls. In addition to antitumor activity we observed an increase in the mouse hematopoietic progenitor cell populations, BFU-e and CFU-GM. These results demonstrated the effects of a short hexameric oligonucleotide telomere mimic in vitro and in vivo and the potential utility of short oligonucleotides as telomerase inhibitors.

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.

The human immunodeficiency virus (HIV) is neuroinvasive and commonly causes cognitive and motor deficits during the later stages of viral infection. (referred to as HIV dementia). The mechanism(s) for disease revolves around secretory products produced from immune-activated brain macrophages/microglia. Recently, we developed an animal model system for HIV dementia that contains xenografts of HIV-1-infected cells inoculated into brains of mice with severe combined immunodeficiency (SCID). This animal system was used to quantitatively evaluate HIV-induced neuropathology. Xenografts of HIV-1-infected human monocytes (placed into the putamen and cortex of SCID mice) remained viable for 5 weeks. HIV-1 p24 antigen expression in mouse brain was persistent. Progressive inflammatory responses (including astrogliosis and cytokine production), which began at 3 days, peaked at day 12. The range of astrocyte proliferative reactions exceeded the inoculation site by > 1000 microns. Brains with virus-infected monocytes showed a > or = 1.6-fold increase in glial fibrillary acidic protein (staining distribution and intensity) as compared with similarly inoculated brains with uninfected control monocytes. These findings paralleled the accumulation and activation of murine microglia (increased branching of cell processes, formation of microglial nodules, interleukin (IL)-1 beta and IL-6 expression). An inflammatory reaction of human monocytes (as defined by HLA-DR, IL-1 beta, IL-6, and tumor necrosis factor-alpha expression) and neuronal injury (apoptosis) also developed after virus-infected monocyte xenograft placement into mouse brain tissue. These data, taken together, demonstrate that this SCID mouse model of HIV-1 neuropathogenesis can reproduce key aspects of disease (virus-infected macrophages, astrocytosis, microglial activation, and neuronal damage). This model may serve as an important means for therapeutic development directed toward improving mental function in HIV-infected subjects with cognitive and motor dysfunction.

The immunologic attributes of cytokine mobilized peripheral blood stem cell (PSC) products (n = 52) and the resulting reconstitution of the hematopoietic and immunologic system following autologous transplantation were examined in a consecutive population of non-Hodgkin lymphoma (NHL), or solid tumor patients at the University of Nebraska Medical Center. Granulocyte-monocyte colony stimulating factor (GM-CSF)-mobilized PSC products had a high frequency of monocytes (31%) and bands (15%) as compared to normal peripheral blood (PB) cells. The phenotypic analysis of the mobilized PSC product revealed that they had normal levels of CD4+ cells, an increased frequency of CD8+ cells and a corresponding decrease in the CD4+:CD8+ cell ratio as compared to the peripheral blood leukocytes (PBL) of normal individuals. PSC products also had an increase in CD34+ cells as compared to PB. Natural killer (NK) and T cell activity in the PSC products were also lower than that observed in PB. Post-transplantation there was an accelerated reconstitution of NK-cell function in the PB as compared to T cell function (PHA (phytohemagglutinin) mitogenesis) which did not return to normal by day 100 post-transplantation. We also report for the first time high levels of an irradiation resistant suppressor cell activity in the PSC product and in the PB post-transplantation. There was also a concomitant increase in CD4-, CD8-, TCR alpha/beta+ cells (phenotypic homolog of 'natural suppressor' (NS) cells) in the PB post-transplantation. The number of months of prior chemotherapy correlated with PHA response but the NS activity and frequency of CD4-, CD8- and TCR alpha/beta+ cells did not. Further, cytokine mobilization and apheresis appears to contribute to the loss of PHA responsiveness and the increased levels of suppressor cell activity.