Expression of Flt3 and c-kit during growth and maturation of human CD34+CD38- cells.

Studies of murine stem cells suggest that the cytokine receptors Flt3 and c-kit are expressed differentially on the earliest reconstitutional cells, such that Flt3 is not expressed until after stem cell activation. Much less is known about the expression of Flt3 and c-kit on primitive human cells, especially those mobilized into circulation for transplantation. In this study, early circulating precursors were analyzed for expression of Flt3 at the gene and protein levels. Flow cytometric studies showed that >90% of CD34+CD38- cells expressed Flt3 antigen (CD135). The proportion of fresh CD34+ cells expressing Flt3 decreased as CD38 staining increased. These results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, which showed that Flt3 gene expression generally was limited to the CD34+CD38- population. Because Flt3 ligand (FL) enhances the growth and/or maintenance of primitive cells, it was important to know how long early cells retain Flt3 receptor expression in expansion culture. Both RT-PCR analyses and functional tests demonstrated that primitive cells are capable of expressing Flt3 for as long as 2 weeks in liquid medium. During the first week of culture, FL enhanced the generation of cells and progenitors without causing a loss of primitive CD34+CD38-Flt3+ cells. Flt3 expression in cell cultures was limited to precursors retaining a CD34+CD38(-/lo) phenotype. Because the most primitive human precursors are believed to express c-kit at a low level, we examined the FL responsiveness of CD34+CD38-c-kit(-/lo) cells and CD34+CD38-c-kit+ cells. CD34+CD38-c-kit(-/lo), cells constituted a small fraction (12%) of the CD34+CD38- population. Whereas both c-kit(-/lo) and c-kit+ subsets were stimulated by FL, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34+CD38-c-kit(-/lo) cells. Furthermore, the rapid response to FL suggests that primitive CD34+CD38-c-kit(-/lo) cells express Flt3 at the time of isolation or shortly thereafter. These results demonstrate the presence of Flt3 on CD34+CD38 blood cells and suggests that Flt3 also may be present on a c-kit(-/lo) subset, among the most primitive in circulation. Flt3 is lost during maturation to committed (CD34+CD38+) lineages. Addition of FL to primitive cell cultures stimulates cell expansion while maintaining early CD34+CD38-Flt3+ precursors for at least 7 days. The possible existence of a more primitive CD34+CD38-c-kit(-/lo) Flt3(-/lo) precursor remains to be determined.

Flt3 ligand enhances the yield of primitive cells after Ex vivo cultivation of CD34+ CD38dim cells and CD34+ CD38dim CD33dim HLA-DR+ cells.

Flt3 ligand (FL) has been proposed as a possible modulator of early hematopoietic cell growth. The purpose of this study was to analyze the impact of FL on ex vivo expansion of hematopoietic cells obtained from adult donors. We sought to precisely identify hematopoietic populations responsive to FL and to quantitate the ability of FL to enhance the survival and/or proliferation of early hematopoietic precursors in a stroma-free culture system. Towards that end, four CD34+ subsets were isolated and their response to FL was characterized. In methylcellulose, FL significantly increased colony formation by CD34+ CD38dim cells but not CD34+ CD38+ cells. In suspension culture, the enhancement of cell expansion by FL was 10 times greater with the CD34+ CD38dim fraction than the CD34+ CD38+ fraction. FL stimulated the generation of colony-forming unit-granulocyte-macrophage (CFU-GM) from the CD34+CD38dim fraction by 14.5- +/- 5.6-fold. To determine if CD34+ CD38dim cells responded uniformly to FL, the population was subdivided into a CD34+ CD38dim CD33dim HLA-DR+ (HLA-DR+) fraction and a CD34+ CD38dim CD33(dim) HLA-DRdim (HLA-DRdim) fraction. FL was far more effective at stimulating cell and progenitor growth from the HLA-DR+ fraction. To determine if FL enhanced or depleted the number of precommitted cells in expansion culture, CD34+ CD38dim and HLA-DR+ fractions were incubated in liquid culture and analyzed by flow cytometry. Inclusion of FL enhanced the absolute number of primitive CD34+ CD33dim cells and CD34+ HLA-DRdim cells after 5 to 12 days of cultivation. To confirm immunophenotypic data, the effect of FL on long-term culture-initiating cells (LTCIC) was determined. After 2 weeks of incubation of CD34+ CD38dim or HLA-DR+ cultures, LTCIC recoveries were significantly higher with FL in 5 of 6 trials (P < . 05). For HLA-DR+ cells, LTCIC recoveries averaged 214% +/- 87% of input with FL and 24% +/- 16% without FL. In contrast, HLA-DRdim LTCIC could not be maintained in stroma-free culture. We conclude that less than 10% of CD34+ cells respond vigorously to FL and that those cells are contained within the HLA-DR+ fraction. FL stimulates the expansion of total cells, CD34+ cells, and CFU-GM and enhances the pool of early CD34+ CD33(dim) cells, CD34+ HLA-DRdim cells, and LTCIC. These data indicate that it is possible to expand hematopoietic progenitors from adult donors without losing precursors from the precommitted cell pool.

The combined effects of interleukin-11, stem cell factor, and granulocyte colony-stimulating factor on newborn rat hematopoiesis: significant enhancement of the absolute neutrophil count.

Dysregulation of neonatal myelopoiesis and thrombopoiesis predisposes the newborn to develop neutropenia and/or thrombocytopenia during states of increased demand. We have previously examined the effects of granulocyte colony-stimulating factor (G-CSF) alone or in combination with either stem cell factor (SCF) or interleukin-11 (IL-11) on in vivo neonatal rat hematopoiesis. In this study, we determined the effect of the triple combination of IL-11, SCF, and G-CSF on newborn rat hematopoiesis. Newborn Sprague-Dawley rats (< or = 24 hours old) were administered intraperitoneal (IP) injections of 250 micrograms/kg IL-11, 100 micrograms/kg SCF, 5 micrograms/kg G-CSF, or various combinations or phosphate-buffered saline (PBS)/human serum albumin (HSA) x 14 d. Platelet and blood cell counts were obtained on days 1, 3, 6, 8, 10, and 13; on day 14 bone marrow neutrophil storage pool (BM NSP), neutrophil proliferative pool (NPP), colony-forming units-granulocyte/macrophage (CFU-GM), and CFU-GM proliferative rates were determined. The triple combination failed to significantly increase the circulating hematocrit over other combinations or placebo. The circulating platelet counts, however, significantly increased during each of the IL-11 treatment arms, but they were not enhanced by the addition of either SCF, G-CSF, or the combination. The triple combination of IL-11, SCF, and G-CSF induced the most significant increase in the circulating absolute neutrophil count (ANC) above any other combination or placebo. Circulating ANC increased 12-fold following the triple combination vs. PBS/HSA (day 14 ANC 16525 +/- 1340 vs. 1368 +/- 197) (p < 0.001). The triple combination of IL-11, SCF, and G-CSF also induced the most significant increase in the BM/CFU-GM proliferative rate and BM NPP, p < 0.002 and p < 0.008, respectively. The highest increase in CFU-GM colony formation, however, occurred with both early lineage CSFs, that is, IL-11 plus SCF, and it was not further enhanced by the addition of G-CSF. These data suggest that the combination of two early-lineage CSFs, IL-11 plus SCF and G-CSF, significantly induces newborn rat myelopoiesis and that IL-11 alone significantly induces newborn rat thrombopoiesis. These results may be helpful in the design of future therapies to treat and/or prevent cytopenias in the newborn.

Effect of interleukin-11 with and without granulocyte colony-stimulating factor on in vivo neonatal rat hematopoiesis: induction of neonatal thrombocytosis by interleukin-11 and synergistic enhancement of neutrophilia by interleukin-11 + granulocyte colony-stimulating factor.

IL-11, a new hematopoietic cytokine isolated from primate stromal cells (PU-34), has been shown to act synergistically with IL-3 to induce proliferation of early hematopoietic stem cells and induce in vitro CFU-MEG proliferation. We hypothesize that recombinant human (rh)IL-11 alone or in combination with granulocyte colony-stimulating factor (G-CSF) might modulate newborn in vivo granulopoiesis and thrombopoiesis. Newborn Sprague-Dawley rats were given 14 d of intraperitoneal rhIL-11 (0-250 micrograms/kg x 14 d), rhIL-11 (250 micrograms/kg) + rhG-CSF (5 micrograms/kg simultaneously x 14 d), rhIL-11 x 7 d followed by G-CSF x 7 d, G-CSF x 14 d, PBS/human serum albumin x 7 d followed by G-CSF x 7 d, or PBS/human serum albumin x 14 d. rhIL-11 alone had no effect on the circulating hematocrit or absolute neutrophil count. There was, however, a significant increase in the circulating platelet count after rhIL-11 (100 and 250 micrograms/kg) versus PBS/human serum albumin (d 13: 1241 +/- 54, 1262 +/- 58 versus 939 +/- 38 k/mm3; p = 0.01). Sequential and simultaneous IL-11 + G-CSF caused a significant increase in the marrow neutrophil reserve and the circulating absolute neutrophil count above that observed when G-CSF alone was administered. IL-11 +/- G-CSF, however, failed to reduce the 96-h mortality rate during experimental group B streptococcal sepsis. These data suggest that IL-11 alone results in a significant elevation in the blood platelet concentration and, in combination with G-CSF, induces an increase in in vivo neonatal rat myelopoiesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro effects of stem cell factor and PIXY321 on myeloid progenitor formation (CFU-GM) from immunomagnetic separated CD34+ cord blood.

Two novel cytokines, stem cell factor (SCF) and PIXY321 (a fusion protein, granulocyte macrophage colony-stimulating factor+IL-3), have recently been demonstrated to enhance in vitro adult myelopoiesis. In this study, we compared the success of separating very early hematopoietic progenitor cells (CD34+) from both cord blood (CB) and adult bone marrow (ABM) and their differential response to SCF, PIXY321, and other later-acting colony-stimulating factors (CSF). Briefly, CD34+ cells were isolated from CB and ABM with an anti-CD34 MAb, HPCA-1, and incubated with various combinations of SCF, PIXY321, and other CSF. The percentage of CD34+ cells was decreased in CB compared to ABM before separation (0.54 versus 1.71%) (p = 0.05). Isolated CD34+ cells from CB and ABM were similar in lineage with respect to CD38, HLA-DR, CD33, and CD5, but decreased in CB with respect to B-lineage expression (CD19, CD10, and CD22) (p = 0.05). SCF increased colony forming unit-granulocyte-macrophage (CFU-GM) formation from CB CD34+ cells compared to unconditioned media and had a significant additive increase with IL-3 (p = 0.006) and granulocyte colony-stimulating factor (p = 0.03). SCF also had an additive increase in CB CFU-GM formation with PIXY321 (p = 0.007). PIXY321 had a similar increase in CFU-GM formation from both CB and ABM CD34+ cells compared to the combination granulocyte macrophage colony-stimulating factor + IL-3. When SCF was added to IL-3, PIXY321, or PIXY321 + IL-6, there was an increase in CFU-GM from CB versus ABM CD34+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of stem cell factor with and without granulocyte colony-stimulating factor on neonatal hematopoiesis: in vivo induction of newborn myelopoiesis and reduction of mortality during experimental group B streptococcal sepsis.

Neonatal hematopoiesis and host defense are developmentally immature and under states of increased demand predispose the newborn to peripheral cytopenias and depletion of bone marrow storage pool reserves. We have previously demonstrated that recombinant human granulocyte colony-stimulating factor (rhG-CSF) can significantly modulate neonatal rat granulopoiesis and act synergistically with antibiotic therapy to reduce the mortality rate during experimental group B streptococcal sepsis. Stem cell factor (SCF) has been shown to stimulate early hematopoietic progenitor cells and, in the presence of lineage-specific CSFs, enhance committed progenitor cell proliferation. In the present study we examined the in vivo neonatal hematologic effects of recombinant rat (rr) SCF (14 days), simultaneous rrSCF + rhG-CSF (14 days), and sequential combination of rrSCF (7 days) + rhG-CSF (7 days). Sprague-Dawley newborn rats (less than or equal to 24 hours) were injected intraperitoneal (IP) x 14 days with the above combinations. rrSCF (0 to 200 micrograms/kg/d) had a negligible effect on the peripheral platelet count and absolute neutrophil count (ANC) but the diminution in the hematocrit during the first 10 days of treatment was less pronounced (P = .0001). However, the simultaneous use of rrSCF + rhG-CSF synergistically increased the circulating day 6 to 13 ANC (P = .001). Similarly, sequential rrSCF + rhG-SCF also had a synergistic significant effect during the second week of therapy on the circulating ANC (P = .01). The bone marrow neutrophil storage and proliferative pools were also significantly increased in newborn rats treated with rrSCF + rhG-CSF versus rhG-CSF (P = .02). The bone marrow and liver/spleen CFU-GM pool was unchanged; however, the CFU-GM proliferative rates were significantly increased in the rrSCF + rhG-CSF group (P = .04). rrSCF also induced a significant increase in the bone marrow and liver/spleen mast cell pool (P = .002). Lastly, rrSCF x 14 days +/- rhG-CSF significantly reduced the mortality rate at 48 and 120 hours after experimental group B streptococcus sepsis (P = .03 and .05, respectively). These data suggest that combination SCF + G-CSF therapy compared with G-CSF alone significantly increases the neonatal rat peripheral neutrophil count, bone marrow myeloid pools and proliferative rates, and induces a reduction in the mortality rate during experimental bacterial sepsis. SCF therapy may have future potential applications in the modulation of human neonatal hematopoiesis and host defense.

Circulating granulocyte colony-stimulating factor (G-CSF) levels after allogeneic and autologous bone marrow transplantation: endogenous G-CSF production correlates with myeloid engraftment.

Myeloid engraftment after bone marrow transplantation (BMT) is influenced by a number of variables, including cytoreductive chemoradiotherapy, genetic disparity, number of reinfused committed myeloid progenitor cells, healthy microenvironment, and the presence of hematopoietic growth factors. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation of myeloid progenitor cells and enhances myeloid engraftment after BMT. We investigated the temporal relationship between endogenous G-CSF production and myeloid engraftment in both children and adults after allogeneic (ALLO) and autologous (AUTO) BMT. Circulating endogenous G-CSF levels ranged between 0 and 2552 pg/mL. The correlation coefficient between circulating serum G-CSF levels and the peripheral absolute neutrophil count (ANC) was r = -.875 (P less than .001). The endogenous serum G-CSF level was highest during the first week after BMT, when the ANC was less than or equal to 200/microL (699 +/- 82.3 pg/mL) (P less than .001). Both children and adults demonstrated a similar inverse relationship between circulating G-CSF level and degree of neutropenia. One patient failed to engraft after AUTO BMT and also failed to generate any endogenous G-CSF production. Lastly, once the serum G-CSF level decreased to less than 200 pg/mL, a mean of 6.1 +/- 0.9 days elapsed before the ANC was greater than or equal to 500/microL for 2 consecutive days. This study demonstrates that endogenous G-CSF production is associated with myeloid engraftment in both children and adults after AUTO and ALLO BMT and that the rate of increase and decrease in endogenous G-CSF may be predictive of either failure to engraft or duration of neutropenia.

In vivo hematological effects of seven days of rhIL-6 in neonatal rats.

Interleukin 6 (IL-6) has been demonstrated to possess a variety of biological and immunological functional properties. Recently, Interleukin 6 has been shown to stimulate in vitro fetal hematopoiesis. In the present study, we investigated the effects of 7 days of rhIL-6 on newborn rat in vivo myelopoiesis. Sprague-Dawley newborn rats (less than 24 h old) were injected (intraperitoneally) daily for 7 days with either rhIL-6 or PBS/BSA. RhIL-6 induced a mild but significant increase in the peripheral neutrophil count on day 1 (P less than 0.02), but had no significant sustained effects during the remaining 7 days of administration. Additionally, rhIL-6 had no significant effect on the bone marrow neutrophil proliferative pool, neutrophil storage pool, the liver/spleen neutrophil storage pool and neutrophil proliferative pools. Although rhIL-6 induced a significant increase in the day 1 platelet count (p less than 0.03), it failed to induce a sustained significant increase in the platelet count during the remaining 7 days of administration. RhIL-6 also failed to induce any change in the circulating hematocrit during the 7 days of administration. This study suggests that rhIL-6 failed to induce a significant and sustained increase in the peripheral neutrophil count or platelet count during in vivo administration in neonatal rats. Although rhIL-6 may possess additional immunomodulating effects in the newborn, this 7-day study of rhIL-6 failed to demonstrate any induction of neonatal peripheral neutrophilia or modulation of neonatal myeloid proliferation.

Sequential administration of interleukin-6 and granulocyte-colony stimulating factor in newborn rats: modulation of newborn granulopoiesis and thrombopoiesis.

During states of increased demand, neonatal host defense is characterized by dysregulation of granulopoiesis, resulting in a high incidence of neutropenia. This study investigated the modulation of neonatal rat hematopoiesis by 14-d administration of recombinant human (rh) IL-6, rh-granulocyte-colony stimulating factor (G-CSF), or sequential combination of rhIL-6 and rhG-CSF. Specifically, newborn Sprague-Dawley rats were treated with either rhIL-6 (5 micrograms/kg/d for 14 d), rhG-CSF (5 micrograms/kg/d for 14 d), rhIL-6 for 7 d followed by rhG-CSF for 7 d, PBS/BSA for 7 d followed by rhG-CSF for 7 d, or PBS/BSA for 14 d. RhIL-6 alone significantly increased the peripheral platelet count during the latter part of the 2nd wk of administration (d 13: 980 +/- 42 versus 716 +/- 23 x 10(3)/mm3) (p = less than 0.001) (mean +/- SEM). Treatment with rhIL-6 for 7 d followed by rhG-CSF significantly increased the peripheral neutrophil count compared with 7 d of PBS/BSA and 7 d of G-CSF (d 14 absolute neutrophil count 4888 +/- 12 versus 2720 +/- 317/mm3) (p = less than 0.05). Similarly, sequential rhIL-6/rhG-CSF significantly increased the d-14 bone marrow neutrophil storage pool (9873 +/- 882 versus 3564 +/- 159/mm3) (p = less than 0.005). Lastly, sequential rhIL-6/rhG-CSF induced the highest increase in bone marrow (p less than 0.01) and liver/spleen CFU-GM pool (p less than 0.001) compared with any other treatment group. These studies suggest that rhIL-6 alone is associated with a significant increase in the neonatal platelet count.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of neonatal myelopoiesis in newborn rats after 7 days' administration of either granulocyte-monocyte colony stimulating factor or interleukin-3.

Single-pulse administration of either recombinant human granulocyte-monocyte colony stimulating factor or recombinant human granulocyte colony stimulating factor to newborn rats has previously been demonstrated to increase the peripheral neutrophil count and modulate bone marrow (BM) neutrophil pools. In our present study, we investigated the effects of 7 d of either recombinant murine granulocyte-monocyte colony stimulating factor (rmGM-CSF) (75 micrograms/kg/d) or recombinant murine IL-3 (rm IL-3) (10 micrograms/kg/d) on newborn rat myelopoiesis. Sprague Dawley newborn rats (greater than or equal to 24 h) were injected (intraperitoneally) daily for 7 d with either rmGM-CSF, rmIL-3, or PBS/BSA. rmGM-CSF induced a significant increase in the peripheral neutrophil count on d 3 (p less than 0.03) and d 7 (p less than 0.001) (75% increase). Additionally, rmGM-CSF induced a 50% increase in the BM neutrophil storage pool (p less than 0.025). rmIL-3 increased the BM colony forming unit-granulocyte monocyte pool (p less than 0.001); however, it failed to increase the peripheral neutrophil count or BM neutrophil storage pool. Neither CSF increased the BM neutrophil proliferative pool or BM colony forming unit-granulocyte monocyte proliferative rate. Additionally, 7 d of rmGM-CSF with or without antibiotics did not synergistically alter the mortality rate after group B streptococcol inoculation. This study suggests that rmIL-3 appears to stimulate more neonatal myeloid committed progenitor cell activity compared with rmGM-CSF. Optimal modulation of neonatal myelopoiesis may require the use of a sequential combination of hematopoietic CSF, namely an early-acting CSF followed by a more lineage myeloid-specific CSF.

Seven-day administration of recombinant human granulocyte colony-stimulating factor to newborn rats: modulation of neonatal neutrophilia, myelopoiesis, and group B Streptococcus sepsis.

Single-pulse administration of rhG-colony-stimulating factor (CSF) to neonatal rats was previously demonstrated to induce peripheral neutrophilia and modulate bone marrow (BM) neutrophil storage and proliferative pools (NSP + NPP). In this study, we investigated the prolonged effects of 7 days of rhG-CSF therapy (5 micrograms/kg/per day). Sprague-Dawley newborn rats (less than or equal to 24 hours) were injected intraperitoneally (IP) (daily for 7 days) with rhG-CSF or phosphate-buffered saline/human serum albumin (PBS/HSA). RhG-CSF induced a significant early and late peripheral neutrophilia: 6,905 +/- 1,625 (day 1) and 9,223 +/- 515 microL (day 7) v 1,275 +/- 90/microL (P less than or equal to .0001). In addition, 7 days of rhG-CSF resulted in a significant increase in the BM NSP: 3,247 +/- 190/microL v 1,677 +/- 339/microL (P less than or equal to .001). There was, however, no depletion or significant change in the BM NPP. Seven days of rhG-CSF also induced a mild increase in BM CFU-GM colony formation (P less than or equal to .01). There was, however, no significant change in liver/spleen CFU-GM colonies or in the CFU-GM proliferative rate in either the BM or liver/spleen cultures. Finally, 7 days of prophylactic rhG-CSF therapy resulted in a synergistic response with antibiotic therapy and significantly modulated the mortality rate during experimental group B streptococcal sepsis (GBS) (100% v 50%) (GvsC) (P less than or equal to .001). Pulse rhG-CSF administered at 6 hours or 18 hours after GBS inoculation, however, failed to act synergistically with antibiotics to improve survival or prevent peripheral neutropenia. This study suggests that 7 days of prophylactic rhG-CSF therapy induces peripheral neutrophilia, myeloid maturation, increases neutrophil BM reserves and also may provide immunologic enhancement of neonatal host defense during experimental GBS in term neonatal rats.

The human LT system. XII. Purification and functional studies of LT and "TNF-like" LT forms from a continuous human T cell line.

A clone of the continuous human T cell line HUT-102, termed YM 1.2, can spontaneously release alpha-LT in vitro. However, when stimulated with phorbol myristic acetate, these cells release other LT forms. These LT forms were purified to homogeneity by DEAE chromatography, column isoelectric focusing, and polyacrylamide gel electrophoresis. One LT form, termed LT-2, is a 79,000 m.w. component in aqueous solution and composed of 21,000 m.w. subunits. This form is immunologically related to macrophage-derived TNF and has a lytic capacity in vitro on K-562, Molt-4F, and Raji cells similar to that described for cytotoxins derived from NK effector cells, termed NK-CF. A second LT form, termed LT-3, is a single 69,000 m.w. peptide which could not be reduced into the smaller subunits. This form expresses antigens in common with both alpha-LT and TNF, because both anti-LT and anti-TNF were required to completely neutralize cell lytic activity in vitro. Functional testing revealed that the LT-3 form is lytic on all continuous cells tested in vitro, including NK-resistant target cells. The LT-3 component appears similar by immunologic, biochemical, and functional criteria to the LT form derived from primary human cytolytic T cells in vitro. At the levels tested, none of these LT-TNF forms had measurable effects on primary fibroblasts in vitro.

Purification to electrophoretic homogeneity of human alpha lymphotoxin from a cloned continuous lymphoblastoid cell line IR 3.4.

The 70-90,000 molecular weight (MW) alpha (alpha) component of the human lymphotoxin (LT) system has been purified to electrophoretic homogeneity. The alpha LT containing supernatants were obtained from a phorbol myristate (PMA) stimulated cloned continuous human B lymphoblastoid cell line IR 3.4. Supernatants were subjected to a biochemical separation scheme that consisted of chromatography on control pore glass beads, DEAE ion-exchange chromatography, lentil-lectin affinity chromatography, and electrophoresis on 7% native preparative polyacrylamide gels. The specific activity of the alpha LT in the final fractions was from 10(7) to 5 X 10(7) units of LT activity/mg protein. Approximately 3 to 5% of the initial alpha LT was recovered in the final fractions and a purification factor of 25,000 to 30,000 fold was required to achieve homogeneity. The alpha LT preparation from preparative PAGE exhibited concident migration of bioactivity and radioactivity on 5 and 7% native PAGE tube gels. Only a single protein peak was observed when the radiolabeled alpha LT was subjected to a two-dimensional SDS-reducing slab gel.

Five-year experience with clean intermittent catheterization in children.

Ninety-eight children have been treated with clean intermittent catheterization (CIC) over a five-year period. Follow-up in 73 of these is presented herin. The complication rate encountered with the technique is low (7 per cent major complications). Only 21 per cent of the patients were able to maintain persistently sterile urine, although no new bouts of pyelonephritis were encountered in the group since our report of three years ago. Six of 44 (14 per cent) refluxing renal units stopped refluxing on CIC, but in two units reflux de novo developed while on treatment. Most patients (79 per cent) demonstrated stable upper tracts on CIC, while 14 per cent showed increased calicectasis with 7 per cent showing improvement. Thirty-one of the patients were less than three years of age when begun on clean intermittent catheterization. We recommend CIC as the treatment of choice in neurogenic vesical dysfunction where total bladder emptying is a problem and advise that it be started as early as possible.

Cryptococcal septicemia associated with urologic instrumentation in a renal allograft recipient.

A case is presented of a renal allograft recipient with 2 episodes of cryptococcal septicemia temporally related to genitourinary manipulation, which preceded the usual signs of meningeal or cutaneous infection. A review of the literature suggests that cryptococcal disease may, occasionally, manifest itself initially in the genitourinary tract. Therefore, we suggest that cryptococcal infection be suspected in the compromised host who has symptoms of cystitis or bladder outlet obstruction during a short period.

Radionuclide measurement of differential glomerular filtration rate.

The authors sought to determine whether radionuclides could provide a reasonable estimate of differential renal function in five normal dogs and six dogs with unilateral segmental renal infarction. Glomerular filtration rate (GFR) of each kidney was measured by the standard technique using constant infusions of 99mTc-DTPA, iothalamate, and creatinine following ureteral catheterization. These results were correlated with total GFR estimated by bolus injection of 99mTc-DTPA and analysis of the plasma 99mTc-DTPA disappearance curve obtained by blood sampling. Differential GFR was then calculated by multiplying the total GFR from double exponential analysis of this curve (DTPA2) by each of three measures of differential function. These include the percent differential uptake of 99mTc-DTPA and 99mTc-DMSA in the posterior projection as well as the geometric mean of 99mTc-DMSA uptake. There were good correlations between differential GFR calculated from iothalamate clearances obtained at ureteral catheterization and all noninvasive methods involving radionuclides and DTPA2 (r = 0.85 - 0.99). Single exponential analysis of the 99mTc-DTPA plasma disappearance curve was less satisfactory. The authors suggest that measurement of total and differential GFR calculated from plasma clearance of 99mTc-DTPA and external counting may be a useful method with potential clinical applications.

Clean intermittent catheterization in children.

The results of the application of clean intermittent catheterization to children are discussed. Of 34 patients only 7 maintained persistently sterile urine and 52 per cent had more than 1 positive culture in the followup period. Complications of the application of this technique are discussed. The major complication rate was 15 per cent and 6 per cent were considered failures of therapy. Only 1 of the 34 patients demonstrated progression of upper tract disease by excretory urography during the followup period. Intermittent catheterization was found to be effective in preserving renal function as well as helping to improve patient social acceptability.