First Report of Phoma herbarum on Tedera (Bituminaria bituminosa var. albomarginata) in Australia.

Tedera (Bituminaria bituminosa (L.) C.H. Stirton var. albomarginata) has been successfully established across the mixed-farming (wheat-sheep) region of Western Australia because this species has remarkable drought tolerance and can survive the dry-summer period with strong retention of green leaf. A leaf spot symptom involving pale brown lesions with distinct dark brown margins had been observed in genetic evaluation plots of tedera at Medina and Mount Barker, Western Australia, and a Phoma sp. was isolated. Single-spore isolations of a typical Phoma sp. isolate were made onto potato dextrose agar and maintained at 20°C, and a representative culture has been lodged in the Western Australian Culture Collection Herbarium maintained at the Department of Agriculture and Food Western Australia (Accession No. WAC13435). Amplification of the internal transcribed spacer (ITS) 1 and ITS2 regions flanking the 5.8S rRNA gene were carried out with universal primers ITS1 and ITS4 according to published protocol (3). The DNA PCR products were sequenced and BLAST analyses was used to compare sequences with those in GenBank. The sequence had 99% nucleotide identity with the corresponding sequence in GenBank for Phoma herbarum. Isolates also showed morphological (e.g., 1) and molecular (e.g., 2) similarities with P. herbarum as described in other reports. The relevant sequence information for a representative isolate has been lodged in GenBank (Accession No. JQ282910). A conidial suspension of 107 conidia ml-1 from a single-spore culture was spray inoculated onto foliage of 6-week-old tedera plants maintained under >90% relative humidity conditions for 72-h postinoculation. Symptoms evident by 10 days postinoculation consisted of pale brown lesions, mostly 1.5 to 4 mm in diameter, which developed a distinct, dark brown margin. Occasional lesions also showed a distinct chlorotic halo extending 1 to 1.5 mm outside the boundary of the lesion. Infection studies were successfully repeated twice and P. herbarum was readily reisolated from infected foliage. No disease was observed on and no P. herbarum were isolated from water-inoculated control plants. Except for a recent published report of P. herbarum on field pea (Pisum sativum L.) (2), this pathogen has only been noted in the Australian Plant Pest Database as occurring on lucerne (Medicago sativa L.) and soybean (Glycine max (L.) Merr.) in Western Australia in 1985 and on a Protea sp. in 1991. To our knowledge, this is the first published report of P. herbarum as a pathogen on tedera in Australia or elsewhere. That P. herbarum occurs on other hosts in Australia and has a wide host range elsewhere together suggest its potential to be a pathogen on a wider range of host genera and species. References: (1) G. L. Kinsey. No. 1501 in: IMI Descriptions of Fungi and Bacteria. 2002. (2) Y. P. Li et al. Plant Dis. 95:1590, 2011. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Interactions of cytoplasmic granules with microtubules in human neutrophils.

Ultrastructural and functional studies of degranulation responses by human neutrophils have suggested that microtubules (MTs) have a role in the intracellular transport of neutrophil granules. We have found that granule-MT complexes can be isolated from disrupted taxol-treated (1.0 microM) neutrophils, visualized by electron microscopy, and quantified in terms of granules per MT length. After incubation of neutrophils with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), granule-MT complex formation was found to be increased two- to threefold. Enhanced binding of granules to MTs was detectable within 30 s of fMLP stimulation and was dependent on the concentration of fMLP. Incubation of cells with dibutyryl cAMP inhibited this fMLP-stimulated granule-MT complex formation in a dose-responsive fashion. These granule-MT interactions could be reproduced in a cell-free system with neutrophil granules isolated by density gradient centrifugation and MTs polymerized from phosphocellulose-purified tubulin. Furthermore, reconstituted granule-MT interactions were found to be modulated by ATPase inhibitors. Sodium orthovanadate increased granule-MT interactions in a concentration-dependent manner, while AMP-PNP, a nonhydrolyzable ATP analogue, and N-ethylmaleimide decreased or eliminated these interactions. In addition, we found that a MT-activated ATPase could be recovered from intact neutrophil granules by salt extraction, and that extracts enriched in this ATPase contained a polypeptide of between 115 and 120 kD which binds ATP and is immunologically related to kinesin. These studies demonstrate that cytoplasmic granules interact with MTs in human neutrophils in a regulated stimulus-responsive manner, and they suggest that such interactions may involve an MT-based, ATPase-dependent, vesicle translocation system as has been demonstrated in other types of cells.

The mobilization and extracellular release of granular enzymes from human leukocytes during phagocytosis.

The importance of granular (lysosomal) enzymes from neutrophils in producing the tissue damage of acute inflammation has been suggested by much indirect and some direct evidence. This study has investigated the kinetics of release and subsequent fate of granular enzymes from phagocytizing human leukocytes The following observations are made: (a) During phagocytosis, the granular enzyme lysozyme is released from leukocytes into the extracellular medium. (b) Release of lysozyme increases as phagocytic challenge increases, but attains a maximum. (c) Release of lysozyme accompanies phagocytosis and is not a delayed event. (d) The lack of release of a nongranular enzyme, lactic dehydrogenase, indicates that cell damage is not a necessary condition of enzyme release. (e) Like lysozyme, beta-glucuronidase is released from phagocytizing leukocytes. Acid alpha-naphthyl phosphatase and cathepsin also appear to be released, but are not found in appreciable amounts in the extracellular medium, in part because of their lability in solution. These results support the concept that extracellular release of granular enzymes may be a useful secretory function of inflammatory leukocytes which becomes damaging to the host in certain circumstances.

Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells.

Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellular fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribution of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation-dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Aggregation responses of neutrophils as well as adhesion responses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-dependent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells.

Role of secretory events in modulating human neutrophil chemotaxis.

The relationship between neutrophil polymorphonuclear leukocyte (PMN) locomotion and the exocytosis of neutrophil cytoplasmic granules was studied by assessing these processes in cells migrating through micropore filters and by measuring the effects of degranulating stimuli on PMN chemotaxis, orientation, adhesiveness, and ability to bind the chemoattractant f-Met-Leu-[3H]Phe. Studies of cells migrating through cellulose nitrate filters indicated that concentrations of f-Met-Leu-Phe optimal for exocytosis were greater than those optimal for chemotaxis and actually inhibited cell migration. In other studies incubation of PMNs with concentrations of secretagogues causing exocytosis of 30% or greater PMN lysozyme increased cell adhesiveness and inhibited chemotaxis. PMNs that had secreted more than 30% lysozyme appeared round, did not orient in a gradient of chemoattractant, and were capable of significantly less f-Met-Leu-[3H]Phe binding than were control cells. The decreased binding of f-Met-Leu-Phe was not associated with hydrolysis of chemotactic peptide by washed cells, although peptide hydrolysis was caused by cell products secreted extracellularly after vigorous exocytosis. In contrast, when only 10--15% cellular lysozyme was released f-Met-Leu-Phe binding was enhanced significantly and there was no depression of chemotaxis. The data indicate limited exocytosis of intracellular granule contents is associated with increased availability of PMN cehmotactic factor receptors. Vigorous exocytosis is associated with inactivation of chemotactic responsiveness related to increase cell adhesiveness, decreased PMN binding of chemotactic factors, and to hydrolysis of chemoattractants by factors secreted extracellularly.

Purine metabolism in myeloid precursor cells during maturation. Studies with the HL-60 cell line.

In studies with the human promyelocytic leukemia cell line HL-60, we defined changes in intermediary purine metabolism that appear to contribute to the regulation of terminal maturation in myeloid cells. When HL-60 cells were exposed to compounds that induce maturation, consistent alterations in purine metabolism were found to occur within 24 h of culture. Perturbation of guanosine nucleotide synthesis and decreases of up to 50% in intracellular guanylate pool sizes were associated with the induced maturation of these cells in response to diverse inducing agents. While immature HL-60 cells were observed to synthesize purine nucleotides by both de novo and salvage pathways, the activity of both pathways decreased in cells induced to mature, although the relative contribution of purine salvage increased. Moreover, incorporation of the salvage pathway precursor, [14C]hypoxanthine from the intermediate, inosine monophosphate (IMP), into guanylates was reduced by approximately 65% in induced HL-60 cells, reflecting decreased activity of both hypoxanthine phosphoribosyltransferase and IMP dehydrogenase. When various inhibitors of IMP dehydrogenase (mycophenolic acid, 3-deazaguanosine, and 2-beta-D-ribofuranosylthiazole-4-carboxamide) were evaluated for their effects upon HL-60 cells, each agent was found to induce the cells to mature morphologically and functionally. Like other inducers, these agents decreased HL-60 cell proliferation and caused the cells to acquire an ability to phagocytose opsonized yeast and reduce nitroblue tetrazolium. Each agent reduced intracellular guanosine nucleotide pool sizes and induced HL-60 cell maturation at micromolar concentrations. These observations suggest that the size of intracellular guanosine nucleotide pools, the biosynthesis of guanosine nucleotides, and the activity of IMP dehydrogenase may be central to the regulation of terminal maturation in myeloid cells.

Effects of levamisole on normal and abnormal leukocyte locomotion.

The anti-helminthic drug levamisole hydrochloride has been reported to stimulate immune responses in humans and experimental animals. We have investigated levamisole effects on human leukocyte locomotion in vitro in studies of neutrophils and mononuclear cells from normal adults, from patients with Chediak-Higashi disease and from patients with the syndrome of hyperimmunoglobulin E, recurrent pyogenic infections, and defective leukocyte chemotaxis. Directed migration (chemotaxis) of neutrophils and mononuclear cells from normal adults and from the hyperimmunoglobulin E syndrome patients, but not from Chediak-Higashi patients, were stimulated by levamisole at concentrations of 0.01-1.0 micronM, with stimulation observed most consistently at 0.1 micronM. These concentrations of drug also increased cyclic GMP levels in mononuclear cells and enhanced hexose monophosphate shunt activity in neutrophils, but did not alter chemotactic factor-induced changes in the surface charge of neutrophils. Other concentrations of levamisole did not affect leukocyte locomotion except for a high concentration (5.0 mM) which stimulated both random and directed leukocyte migration. When patients with the hyperimmunoglobulin E syndrome took levamisole by mouth, the abnormal chemotactic responses of their neutrophils were significantly improved towards normal. These studies are the first to show pharmacologic improvement of in vitro leukocyte locomotion in patients in whom recurrent infections have been attributed to a defect of this leukocyte function.

Abnormal responses of myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor in human cyclic neutropenia.

Granulocyte-macrophage progenitors (CFU-GM) from four patients with childhood onset cyclic neutropenia demonstrated abnormal in vitro proliferative responses to purified, recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) when examined in detailed dose-response studies. Marrow aspirate specimens were obtained for these studies from cyclic neutropenia patients (both during neutropenic nadirs and during recovery phases of cycles), from leukemia patients in remission who had received myelosuppressive chemotherapy, and from healthy normal volunteers. Nucleated marrow cells were then isolated by density-gradient centrifugation and cryopreserved to permit studies of CFU-GM from patients and controls to be carried out at the same time and in replicate. Maximum clonal growth of CFU-GM from normal subjects and from individuals recovering from drug-induced myelosuppression was elicited by 20-100 pmol/liter rhGM-CSF, and the CSF concentrations that induced half-maximal responses (ED50) were between 1.0 and 3.0 pmol/liter. In contrast, maximum growth of CFU-GM from the cyclic neutropenia patients required greater than or equal to 1.0 nmol/liter rhGM-CSF and ED50's were greater than 30.0 pmol/liter. These abnormalities in the GM-CSF responsive growth of myeloid progenitors were independent of cycle time and were most apparent with the predominantly neutrophilic 7-d CFU-GM. Moreover, differences in the growth of 14-d CFU-GM could be attributed mostly if not entirely to differences in the generation of neutrophilic colonies. These findings indicate that childhood onset cyclic neutropenia is associated with an underlying disturbance in the GM-CSF responsive growth of myeloid progenitors committed to neutrophilic differentiation.

Neutrophil-mediated endothelial injury in vitro mechanisms of cell detachment.

Neutrophil-mediated endothelial injury was assessed in vitro using assays of cell lysis and cell detachment. Activation of human peripheral blood neutrophils adherent to human umbilical vein endothelial cell monolayers by serum-treated zymosan produced dose-dependent endothelial cell detachment without concomitant cell lysis. This injury was inhibited by neutral protease inhibitors, but not by catalase or superoxide dismutase. Neutrophils from a patient with chronic granulomatous disease also produced endothelial cell detachment when activated by serum-treated zymosan similar to normal neutrophils. Endothelial detachment was also produced by cell-free postsecretory media from activated neutrophils or by partially purified human neutrophil granule fraction and was inhibitable by tryptic, elastase, and serine protease inhibitors, but not by an acid protease inhibitor. Analysis of iodinated endothelial cell surface proteins that had been exposed to partially purified neutrophil granule fraction showed complete loss of proteins migrating in the region of fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This result was prevented in the presence of neutral protease inhibitors. We conclude that neutrophil-derived neutral proteases mediate endothelial cell detachment in vitro through digestion of endothelial cell surface proteins including fibronectin.

Exploring the feasibility of selective depletion of T lymphocyte subsets by whole blood immunoadsorption cytapheresis.

Normal turnover of T lymphocytes is slow relative to other blood cells. Consequently, the physical removal of circulating leucocytes by thoracic duct drainage, repeated leukapheresis or blood filtration results in T cell depletion and immunosuppression. However, clinical use of such procedures is impractical compared with immunosuppressive drugs or radiation. None the less, immunosuppression by physical depletion of T cells, avoiding the systemic toxicities of drugs and radiation, might have clinical advantages if immunophenotypically distinct T cell subsets could be depleted selectively. Recent advances in targeted plasma protein apheresis using adsorbent macrobead columns prompted us to determine whether analogous techniques might permit CD4+ T lymphocytes to be removed selectively from whole blood. To explore this possibility, we linked murine anti-human-CD4 and isotype-identical control monoclonal antibodies (mAbs) to agarose, polyacrylamide and polystyrene macrobeads (150-350 microm) and then evaluated the selectivity, specificity and efficiency of macrobead columns to remove CD4+ T cells from anti-coagulated whole blood at varying mAb densities and flow rates. We also examined saturation kinetics and Fc-oriention versus random coupling of mAbs to macrobeads. Sepharose 6MB macrobead (250-350 microm) columns proved to be most effective, selectively removing up to 98% of CD4+ T cells from whole blood. Moreover, depletion efficiency and selectivity were retained when these columns were reused after elution of adherent CD4+ cells. These studies indicate that selective depletion of T lymphocyte subsets by whole blood immunoadsorption apheresis using mAb-linked macrobead columns may be feasible on a clinical scale. It is possible that such apheresis techniques could achieve targeted forms of immunosuppression not possible with drugs or radiation.

Tandem cycles of high-dose melphalan and autologous stem cell transplantation increases the response rate in AL amyloidosis.

Clinical outcomes of patients with AL amyloidosis treated with high-dose melphalan and stem cell transplantation (HDM/SCT) are tightly linked to the achievement of a hematologic complete response (HCR). We conducted a prospective trial to determine whether a second cycle of HDM/SCT could induce HCR in patients in whom the plasma cell dyscrasia persisted following initial treatment with HDM/SCT. Sixty-two patients were enrolled. Nine patients (15%) were removed from the protocol. Of the 53 patients continuing in this study, four died within 100 days of treatment (8%), and 27 (55%) achieved an HCR at 6 months after the first cycle of HDM/SCT. Of the 22 patients who did not achieve an HCR after initial treatment, 17 received a second HDM/SCT, 1 died within 100 days of treatment (6%), while 5 (31%) achieved an HCR. Thus, the HCR rate was 67% (32/48) for surviving patients on study, 60% (32/53) for all patients who received initial cycle of HDM/SCT, and 56% (35/62) by intention-to-treat. The median survival for all patients enrolled on the trial has not yet been reached. Thus, tandem cycles of HDM/SCT can increase the proportion of patients who achieve an HCR.

Clinical and molecular characteristics of patients with non-amyloid light chain deposition disorders, and outcome following treatment with high-dose melphalan and autologous stem cell transplantation.

Light chain deposition disease (LCDD) is caused by a clonal plasma cell disorder in which fragments of monoclonal immunoglobulin light chains form non-fibrillary deposits in various tissues resulting in organ dysfunction. Crystal storing histiocytosis (CSH) is another light chain deposition disorder in which monoclonal light chains form intracytoplasmic crystals. Both are uncommon diseases for which there is limited treatment experience. Between 2003 and 2005, five patients with LCDD and one with CSH were treated at Boston University Medical Center with high-dose melphalan and autologous peripheral blood stem cell transplantation (HDM/SCT). Five of the six patients had predominantly renal involvement, and one patient with LCDD had biopsy-proven deposits in the myocardium. Molecular characterization revealed that the pathologic light chains were kappa in four of the six patients, and sequence analysis revealed unusual germline donor genes and high rates of amino-acid substitutions. One light chain sequence encoded a new potential N-linked glycosylation site, and another showed evidence of antigen selection. All patients are alive and five of the six patients are in complete hematologic remission at a median follow-up of 12 months (range 4-29 months) after HDM/SCT. In our experience, HDM/SCT is a feasible and effective treatment approach for these disorders.

Direct analysis of differentiation proteins in normal and leukemic human granulocytes by high-performance liquid chromatography.

The feasibility of the use of reverse-phase high-performance liquid chromatography (RP-HPLC) for detecting differences in the protein phenotypes of highly purified fractions of normal and chronic myelogenous leukemic (CML) mature granulocytes isolated from peripheral blood was determined. At least 21 protein peaks were consistently and reproducibly detected in the RP-HPLC profiles of acetonitrile-trifluoroacetic acid extracts of normal granulocytes. This assay takes only 60 minutes to perform and can be done on 4 X 10(6) granulocytes (approximately the number of granulocytes in 1 ml normal blood). Furthermore, sodium dodecyl sulfate-gel electrophoresis of the RP-HPLC fractions provides a second dimension to the analysis of the polypeptide pattern of these cell lysates. Analyses of subcellular fractions by these methods revealed that most of the major peaks in the RP-HPLC profiles of intact granulocytes originate mainly from the granule and membrane fractions. Although the protein phenotypes of mature granulocytes were remarkably uniform among normal individuals, those of mature granulocytes obtained from the blood of CML patients were consistently abnormal and varied considerably among individual patients. The results indicate that the approach used here could have useful application in the study of abnormal granulocyte differentiation in leukemia.

Serum free light-chain responses after high-dose intravenous melphalan and autologous stem cell transplantation for AL (primary) amyloidosis.

Serum free light-chain (FLC) concentrations were measured by a sensitive nephelometric immunoassay in 66 patients with AL amyloidosis before and after treatment with high-dose melphalan and autologous stem cell transplantation (HDM/SCT). At 1 year after HDM/SCT, 27 patients (41%) achieved a complete hematologic response (CR), that is, disappearance of the monoclonal gammopathy previously evident by immunofixation electrophoresis (IFE) in serum and urine and of plasma cell clonality in the bone marrow. Abnormally elevated FLC levels became normal in 27 patients (41%), and decreased by >90% in 37 (56%). Average improvements in FLC were 94% for patients who achieved a CR and 72% for those who did not (P=0.0001). However, a reduction in FLC of >90% was associated with a similar high likelihood of clinical improvement and prolonged survival, whether or not patients achieved a CR. While CR, as defined by standard criteria, is a more stringent indicator of hematologic response than are decreases in abnormally elevated FLC levels per se, these measures of hematologic response are complementary, and decreases in FLC are more readily detected early after treatment than are the changes in IFE and marrow studies required to determine CR.

Predictive factors for hematopoietic engraftment after autologous peripheral blood stem cell transplantation for AL amyloidosis.

Treatment of patients with AL amyloidosis with high-dose melphalan and autologous peripheral blood stem cells (PBSC) produces hematologic remissions in approximately 40% of evaluable patients, accompanied by improvements in organ disease and quality of life. These patients, who frequently have amyloid deposits in bone marrow blood vessels and interstitium and impaired function of kidneys, liver, spleen, and heart, represent an unusual population for stem cell transplantation, with unique problems. To identify factors influencing engraftment rates after chemotherapy and autologous granulocyte colony-stimulating factor (G-CSF)-mobilized PBSC reinfusion, we studied a group of 225 patients. The median time to neutrophil engraftment was 10 days (range, 8-17 days). In a multivariate analysis, the factors positively affecting the rate of neutrophil engraftment were CD34+ stem cell dose, female gender, and minimal prior alkylator therapy. The median time to platelet engraftment was 13 days (range, 7-52 days). Factors positively affecting platelet engraftment, in addition to CD34+ cell dose, included preserved renal function and the absence of neutropenic fever. The conditioning dose of intravenous melphalan was not found to be an independent predictive factor for hematopoietic recovery. Thus, in this patient population, organ function and host and hematopoietic factors influence engraftment after PBSC rescue.

Affinity purification and subcellular localization of kinesin in human neutrophils.

Studies of granule-microtubule interactions in human neutrophils have suggested that mechanochemical ATPases such as kinesin or dynein may play a role in granule mobilization during neutrophil activation by inflammatory signals. In this study we show that proteins extracted from the surface of neutrophil granules, found previously to contain microtubule-dependent ATPase activity, caused microtubules polymerized from phosphocellulose-purified rat brain tubulin to move across glass slides. Antibodies were generated against peptides based on two regions of the amino acid sequence of Drosophila kinesin: the ATPase active site (amino acids 86-99) in the head of the kinesin heavy chain and the tail of the heavy chain (residues 913-933). These antibodies were found to recognize kinesin in rat brain extracts as well as kinesin-like polypeptides in extracts of human neutrophils. Furthermore, when used in immunoaffinity chromatography, these antibodies permitted the isolation of a protein from neutrophil granule extracts that was recognized by Drosophila kinesin antibodies. Subcellular localization by immunofluorescence microscopy showed this protein to be associated principally with the cytoplasmic granules of neutrophils.

Anti-CD34+ Fabs generated against hematopoietic stem cells in HIV-derived combinatorial immunoglobulin library suggest antigen-selected autoantibodies.

Bone marrow suppression associated with HIV infection does not appear to be solely due to direct viral cytopathic effects. Autoantibodies may play a role in myelosuppression, however it is unclear whether autoantibodies produced in HIV infection represent a primary pathogenic process or merely reflect polyclonal B cell activation. To address these questions, we generated combinatorial immunoglobulin libraries using the pComb3 phagemid from an HIV+ individual with evidence of circulating autoantibodies. From one library, three anti-CD34 Fabs were identified using fresh CD34+ cells as antigenic targets by a method of phage subtraction. The anti-CD34 Fabs are specific by immunoblotting and Elisa and are of high affinity, with calculated Kds in the range of 10(-7) -10(-8) M. Nucleic acid sequencing revealed all three to be of the VH3 family and to have lambda light chains with some gene segments expressing little somatic mutation, while other segments were somatically mutated in patterns suggestive of antigen selection. These findings indicate that (1) A subset of HIV-associated anti-CD34 autoantibodies are monospecific and antigen-selected and are not merely a consequence of polyclonal B cell activation and elevated Ig levels in HIV. Autoreactivity in HIV therefore includes both polyspecific, low affinity antibodies as well as monospecific antigen-selected high affinity antibodies. (2) Although bone marrow suppression in HIV is likely to be multifactorial, autoantibodies to hematopoietic stem cells may contribute to its pathogenesis. (3) Library sampling of VH gene family rearrangements shows no evidence for under-representation of the VH3 family in the immune dysregulation of HIV infection. Phage subtraction is corroborated to be an effective means of identifying, cloning, and characterizing antibodies to hematopoietic differentiation antigens.

Effects of anti-CD33 blocked ricin immunotoxin on the capacity of CD34+ human marrow cells to establish in vitro hematopoiesis in long-term marrow cultures.

Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.

Efficiency of human HLA-mismatched CD34+ cells from unrelated donors in establishing in vitro hematopoiesis in allogeneic long-term marrow cultures.

We have examined the capacity of highly purified human CD34+ marrow cell isolates from unrelated, HLA-mismatched donors to establish in vitro hematopoiesis on recipient marrow stromal cells in 2-stage hematopoietic long-term marrow cultures (H-LTMC). HLA-typing of both peripheral blood mononuclear cells and CD34+ marrow cells was performed for both HLA class I and HLA class II antigens for eight healthy individuals. Significant antigenic mismatches for these molecules ranged from three to six antigens for each recipient-donor pair. Comparison of MHC antigen expression by peripheral blood cells and CD34+ marrow cell isolates confirmed the presence of identical HLA-A, -B, and -C, and -DR specificities on the surface of these cells. Typing of -DQ specificities, however, was not consistently reactive on CD34+ cells. The > or = 20% plating efficiency of purified CD34+ cells for BFU-E, CFU-GM, and CFU-MIX allowed us to use inoculum doses of 10(3), 10(4), and 10(5) cells to determine the efficiency of allogeneic CD34+ cells in achieving in vitro engraftment and the establishment of hematopoiesis in H-LTMC. Engraftment of adherent BFU-E, CFU-GM, and CFU-MIX was equally efficient for autologous and allogeneic CD34+ cells. In vitro hematopoiesis reflected by the cumulative recoveries of progenitor cells over time was also equivalent for allogeneic and autologous CD34+ cells. These results demonstrate that highly purified, HLA-mismatched CD34+ marrow cells proliferate and establish in vitro hematopoiesis as efficiently as autologous cells in marrow derived stromal cell cultures and confirm that interactions between stromal cells and highly purified CD34+, DR-, and CD34+, DR+ marrow cell isolates are not MHC-restricted.

Inhibitory effects of HIV-1-infected stromal cell layers on the production of myeloid progenitor cells in human long-term bone marrow cultures.

This report presents the results of studies using long-term bone marrow cultures (LTBMC) of human bone marrow cells to investigate the effect of HIV-1 on in vitro hematopoiesis. Confluent stromal cell layers established from human bone marrow cells were irradiated to eliminate residual hematopoietic progenitor cells and exposed to HIV-1ADA or to HIV-1IIIB, monocytotropic and lymphocytotropic strains of HIV-1, respectively. A productive infection did not develop in cultures exposed to HIV-1IIIB but did for cultures exposed to HIV-1ADA as there was a progressive increase in HIV-1 p24 antigen. Stromal cell layers infected with HIV-1ADA were also cocultured with autologous CD34+ bone marrow cells. Four days, 1, 2, and 3 weeks later, the number of colony-forming units granulocyte/macrophage (CFU-GM) in non- and HIV-infected LTBMC was determined. The number of CFU-GM increased during the first week in both non- and HIV-infected LTBMC. One week after the coculture of CD34+ cells with stromal cell layers infected with HIV-1ADA, the number of CFU-GM in six out of eight experiments was reduced compared to noninfected control LTBMC. In those six experiments, the number of CFU-GM was 53 +/- 6% standard error of the mean (SEM) of the number in noninfected LTBMC. A reduced number of CFU-GM was observed in the nonadherent fraction of HIV-infected LTBMC for at least 2 weeks. These results demonstrate that some cells in the stromal cell layers of LTBMC were targets for HIV-1 and that HIV-infected stromal cell layers suppressed or delayed the production of CFU-GM.