Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow

Natural killer (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK- LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the "lymphoid" gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5- fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500.     

Interleukin-2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients: II. Feasibility of LAK generation in children with active disease and in remission

Activation and expansion in culture with rIL-2 of peripheral blood (PB) and/or bone marrow (BM) specimens derived from children with ALL and ANLL, with active disease (AP) and in remission were studied (RP). Baseline NK cytolytic activity from AP was found to be depressed, whereas RP-derived cells had normal NK activity, as assayed against K562 targets. Culture in rIL-2 significantly enhanced the NK activity of both AP- and RP-derived cells and generated LAK activity, as assayed by 4-hour 51Cr release, against NK-resistant Raji cell line and against fresh, allogeneic, and autologous tumor cells. Lytic activity against fresh, cryopreserved leukemia blasts was of lower than that found against cell lines. In three patients higher lytic activity against autologous than against allogeneic blasts was demonstrated. Expansion in culture with rIL-2 varied from twofold to 120-fold. rIL-2 activation and expansion was better in RP than in AP. The predominant phenotype of activated cells, as determined by flow cytometry, was [mean % (SD)]: CD3- = 54 (12), CD8+ = 55 (17), and NKH1+ = 26 (7). The consistently high level of CD8+ cells was accompanied by very low levels of CD4+ cells: mean = 11% (14). Double-marker analysis showed mean of 33% (10) for CD3+/NKH1+ cells and mean = 32 (11) for CD8+/NKH1+ cells, implying that these populations were overlapping. Kinetics of expression of cell surface markers during 2 to 3 weeks in culture showed that CD8+ and NKH1+ enrichment occurred during the first week and lasted for up to 4 weeks, whereas CD4+ expression decreased after the second week. A significant decrease in the expression of IL-2 receptors (CD25) was observed from the second week of culture. This study shows the feasibility of in vitro generation of killer cells from PB and BM of pediatric leukemia patients.     

Effect of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on hematopoietic regeneration as demonstrated in a nonhuman primate chemotherapy model

Using a recently developed hepsulfam-induced pancytopenia model in rhesus macaques, we have studied the effects of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on marrow regeneration. Control animals were given hepsulfam (1.5 g/m2 by a single 30-minute intravenous [i.v.] injection, n = 4), while study animals received hepsulfam followed by rhIL-6, rhIL-3, or a combination of rhIL-6 and rhIL-3 (n = 3 per study group). Each cytokine was administered by once- daily subcutaneous (SC) injection (15 micrograms/kg/d) for 3 weeks beginning the day after chemotherapy (days 2 through 22). Mean platelet counts in control animals were < 100,000/microL on days 15 through 24, with 50% of the counts < 50,000/microL and two of four animals requiring platelet transfusion. In the rhIL-6- and rhIL-6/rhIL-3- treated groups, the nadir mean platelet counts were 164,000 +/- 58,700/microL and 162,300 +/- 23,800/microL, respectively, and occurred on day 15. Platelet counts in the rhIL-3-treated group were similar to those in controls. Mean absolute neutrophil counts (ANCs) < 1,000/microL occurred on days 10 through 29 in control animals, days 8 through 15 in rhIL-6-treated animals, and days 6 through 8 and 13 in rhIL-6/rhIL-3-treated animals. The frequency of ANCs < 500/microL was significantly less in the rhIL-6- and rhIL-6/rhIL-3-treated groups versus control groups (2.7 +/- 0.6 and 2.0 +/- 1.0 vs 7.0 +/- 1.4 occurrences, respectively; P < .05). rhIL-3-treated animals had ANCs similar to those in controls; one animal died with septicemia on day 21. Monkeys receiving rhIL-6 were significantly more anemic during the cytokine administration period; however, the anemia resolved by day 24. Coadministration of rhIL-3 and rhIL-6 partially corrected the anemia. The data indicate that rhIL-6 prevents significant thrombocytopenia and shortens the neutropenic period in this chemotherapy model.     

Interleukin 2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients. I. Feasibility of LAK generation in adult patients with active disease and in remission

The feasibility of in vitro interleukin 2 (IL-2) activation and expansion of mononuclear cells (MNCs) derived from adult patients with acute myelogenous leukemia (ANLL) was studied. Patients' natural killer (NK) and lymphokine-activated killer (LAK) cell activity was compared with that of normal donors in terms of: (a) cytolytic activity (four- hour 51Cr release assay) against an NK-sensitive target (K562), NK- resistant targets (Raji/Daudi), and fresh/cryopreserved autologous and allogeneic leukemic blasts; (b) proliferation and expansion in culture with 1,000 U/mL recombinant IL 2 (rIL 2); and (c) the cell surface phenotype of the cultured cells. In 21 of 24 patients with active disease (AP) MNCs derived from the peripheral blood (PBL) or bone marrow (BM) could be cultured and expanded in the presence of rIL 2. These cultures initially contained between 30% and 50% blasts, and during 2 to 4 weeks of culture destruction of blasts and enrichment of up to 60% in cells with the morphology of large granular lymphocytes (LGLs) was observed. Expansion in culture varied between two- and 100- fold. MNCs from all patients in remission (RP) could be activated by rIL 2 and expanded up to 30-fold after 1 to 3 weeks in culture. NK activity of fresh PBLs from AP was significantly lower than in normal controls, whereas NK activity of RP was within the normal range. High levels of postactivation NK and LAK activity on K562/Raji/Daudi and on fresh/cryopreserved leukemic blasts was generated in approximately 50% of cases of AP and in most RP. Cell surface phenotype studies showed that cultured cells derived from ANLL patients were significantly enriched (up to 40%) in NKH-1 (Leu 19) positive cells, with RP LAK cells also expressing a high proportion of CD16 positive cells (up to 40%). This study has shown that it is feasible to activate and significantly expand killer cells derived from active disease and remission ANLL patients during 1 to 3 weeks culture with IL 2 with good maintenance of cytolytic activity. Both initial NK activity and LAK generation was optimal in remission patients. Based on data from this study, a clinical protocol has been developed for treatment of early relapse ANLL patients with LAK cells cultured for 1 to 3 weeks and systemic IL 2.     

Spectrin-alpha I/61: a new structural variant of alpha-spectrin in a double-heterozygous form of hereditary pyropoikilocytosis

Recent biochemical studies have led to the identification of abnormal spectrins in the erythrocytes of patients with hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis (HE). In this report we describe the biochemical characterization of the erythrocytes from a proband with severe HPP who is doubly heterozygous for two mutant spectrins (Sp): Sp alpha I/74 and a new, previously undetected, mutant of alpha-spectrin designated Sp alpha I/61. The proband's erythrocytes are unstable when exposed to 45 degrees C, and her membrane skeletons exhibit instability to shear stress. The content of spectrin in the proband's erythrocyte membranes is decreased to 75% of control values. The amount of spectrin dimers in crude 4 degrees C spectrin extracts is increased (58%) as compared with control values (6% +/- 4%). Limited tryptic digestion reveals a marked decrease in the normal 80,000-dalton alpha I domain, an increase in the 74,000-dalton fragment that is characteristic of Sp alpha I/74, and an increase in a series of new fragments of 61,000, 55,000, 21,000, and 16,000 daltons. Both parents are asymptomatic, but they have increased amounts of spectrin dimers (17% to 25%). Limited tryptic digestion of the father's spectrin demonstrates the presence of a previously identified abnormal spectrin (Sp alpha I/74) that is characterized by a decrease in content of the 80,000-dalton peptide and an increase in concentration of the 74,000-dalton peptide. The mother's spectrin digests show a decrease in the amount of 80,000-dalton peptide and the formation of new peptides of 61,000, 55,000, 21,000, and 16,000 daltons. The data indicate that this severe form of HPP is due to the inheritance of two distinct abnormal spectrins, Sp alpha I/74 and a new spectrin mutant, Sp alpha I/61.     

Treatment of refractoriness to platelet transfusion by protein A column therapy

Ten thrombocytopenic patients (platelets < 10–24 × 10(9)/L) who were refractory to platelet transfusion were investigated for their responsiveness to staphylococcal protein A column therapy. Nine patients had previously been treated with steroids, intravenous immune globulin, and/or other forms of immunosuppressive therapy without improvement in their transfusion response. All patients were receiving multiple platelet transfusions without achieving 1-hour corrected count increments (CCIs) > or = 7500. Eight patients had antibodies that reacted with platelets and were directed against HLA class I antigens, ABO antigens, and/or platelet-specific alloantigens. Plasma (500-2000 mL) from each patient was passed over a protein A silica gel column and then returned to the patient. Patients received from 1 to 14 treatments. A positive response to protein A therapy was defined as at least a doubling of the pretreatment platelet count and/or two successive 10- to 120-minute posttransfusion CCIs > or = 7500. Following plasma treatments, 6 of 10 patients responded with daily platelet counts that averaged 48 +/− 11 × 10(9) per L as compared with counts of 16 +/− 7 × 10(9) per L (p < 0.0005) before treatment. Posttransfusion CCI values determined in four of these patients averaged 2480 +/− 810 and 10,010 +/− 3540 (p < 0.005) before and after treatment, respectively. In contrast, among the four unresponsive patients, platelet counts averaged 10 +/− 9 and 13 +/− 10 × 10(9) per L (p = NS), respectively, while posttransfusion CCIs were 700 +/− 1410 and 1520 +/− 2460 (p = NS), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfusion-associated graft-versus-host disease in immunocompetent patients: report of a fatal case associated with transfusion of blood from a second-degree relative, and a survey of predisposing factors

A patient without evident immune deficiency who received a transfusion of blood from a second-degree family member developed fatal transfusion- associated graft-versus-host disease (TA-GVHD). The donor was homozygous for an HLA haplotype for which the recipient was heterozygous (one-way HLA match). All 39 reported cases of TA-GVHD in immunocompetent patients were reviewed to ascertain the predisposing factors and to define the indications for irradiating blood for this population. HLA typing was described in 15 cases; in 13, including seven related and six unrelated donors, a one-way HLA match was present. Thirty-one (79%) of the 39 cases were reported from Japan (and 196 other cases are cited in the Japanese literature), but a one-way HLA match among unrelated donors at HLA-A, -B, -DR loci is only approximately two to four times more likely in Japanese persons than in whites. Fresh blood (< 96 hours old) was used in 29 (94%) of the 31 cases reported from Japan and in 33 (87%) of 38 cases overall (in one case, the age of the blood used was not reported). Thus, factors that appear to predispose to TA-GVHD in immunocompetent patients are a one- way HLA match, fresh blood, and, possibly, Japanese ancestry. Irradiating cellular blood components from all blood relatives of transfusion recipients will not completely eliminate the risk of TA- GVHD.     

Establishment of erythropoiesis following bone marrow transplantation in a patient with congenital hypoplastic anemia (Diamond-Blackfan syndrome)

Marrow transplantation was attempted in a 13-yr-old boy with congenital hypoplastic anemia who had never responded to corticosteroid therapy. Prior to the transplant, he had received 238 transfusions, at least 12 of which were from his father. He was prepared for grafting with antilymphocyte globulin, procarbazine, and total body irradiation (1000 rads). The patient, whose red cells were Group B, then received marrow cells from his Group O, histocompatible, sister. Thereafter, reticulocytes, Group O erythrocytes, and female leukocytes appeared in the peripheral blood. Erythroid precursors were seen in the patient's marrow for the first time in his life, and all lacked fluorescent Y chromosomes. Dividing cells were all female. After initially progressing well, the patient developed interstitial pneumonia and died 55 days after the transplant. The successful erythroid graft suggested that this patient's failure to produce red blood cells was due to a defective stem cell rather than to a humoral defect, plasma inhibitor, or abnormal marrow microenvironment. It suggested further that sibling marrow may be engrafted in patients who have received multiple transfusions, even from a parent.     

Psoriatic arthritis – what the dermatologist needs to know

Psoriasis is commonly associated with a co‐existent arthritis known as psoriatic arthritis (PsA). Although there is some treatment overlap for psoriasis and psoriatic arthritis, it is possible that dermatologists may not diagnose or treat appropriately patients who are developing psoriatic arthritis at an early stage of the disease process when joint damage may be preventable. In this article we review the criteria for diagnosis of this sero‐negative arthritis, look at the clinical indications for referral to a rheumatologist and discuss evolving treatment options relevant to both conditions.