A novel single-cell proliferation assay shows that long-term culture- initiating cell (LTC-IC) maintenance over time results from the extensive proliferation of a small fraction of LTC-IC

We have previously shown that when adult marrow CD34+/HLA-DR- cells are cultured for 5 or 8 weeks in the presence of stroma-conditioned media with interleukin-3 (IL-3) and macrophage inflammatory protein-1 alpha (MIP-1 alpha), long-term culture-initiating cells (LTC-IC) are maintained but not expanded. However, if the same cultures are evaluated after 2 weeks, we show that LTC-IC expand 5.5- +/- 0.2-fold. Because expansion of LTC-IC is likely the result of a balance between proliferation and loss of LTC-IC, we hypothesized that, although LTC-IC proliferate in these cultures, loss of a fraction of LTC-IC underlies the lack of long-term expansion. To evaluate the fate of LTC-IC (proliferation, conservation, or loss), we performed PKH-26 labeling assays and developed a single LTC-IC proliferation assay. For PKH-26 labeling assays, CD34+/HLA-DR- cells were incubated with the membrane intercallating dye, PKH-26, before culture for 14 days in stroma- noncontact cultures + IL-3 + MIP-1 alpha. Progeny was reselected by fluorescence-activated cell sorting based on their PKH-26 fluorescence intensity. These studies showed that LTC-IC proliferate because 80% of LTC-IC at week 2 had 0.5 to 1 log lower fluorescence intensity than did freshly labeled CD34+/HLA-DR- cells. To further determine the fate of LTC-IC, we also developed a single LTC-IC proliferation assay. A population of CD34+/CD33- cells, highly enriched in LTC-IC, was sorted singly in stroma-conditioned media+IL-3 + MIP-1 alpha. After 5 weeks, the content of each well was divided equally over 8 secondary stroma- containing wells and cultured for 8 weeks to determine the capacity of the single-cell progeny to initiate 1 or more secondary stromal cultures. Progeny of single-sorted cells were able to initiate up to 8 secondary long-term cultures, demonstrating that LTC-IC proliferate in stroma-conditioned media+IL-3 + MIP-1 alpha. However, more than 65% of single-sorted LTC-IC were not conserved because their progeny could no longer initiate secondary long-term cultures. This finding indicates that, although stromal factors and IL-3 + MIP-1 alpha can induce proliferation of LTC-IC, failure to conserve a large fraction of LTC-IC results in lack of long-term expansion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of granulocyte-macrophage colony-stimulating factor on lymphokine-activated killer cell induction

The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti- IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)- induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.     

Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host disease in H-2- incompatible transplanted SCID mice

In a previous study, we found that total body irradiation (TBI) was essential to induce acute graft-versus-host disease (GVHD) after allogeneic H-2-incompatible splenocyte (SP) transplantation in SCID mice. SCID mice (H-2d) conditioned with cyclophosphamide and transplanted intravenously (IV) with 5 x 10(7) C57BL/6 (H-2b) SP developed chronic GVHD within 3 months posttransplant without any evidence of preceding acute GVHD. In this study, SCID mice were conditioned with 4 Gy TBI or non-TBI regimens, either BuCy2 (busulfan 4 mg/kg/d + cyclophosphamide 100 mg/kg/d for 2 days) or Cy5 (cyclophosphamide 100 mg/kg/d for 5 days), and then transplanted IV with 5 x 10(7) SP. The TBI-conditioned mice were further divided into tree transplant groups: (1) TBI and SP administered the same day (TBI + D0 SP), (2) SP administered 4 days post-TBI (TBI + D4 SP), and (3) SP administered 7 days post-TBI (TBI + D7 SP). The severity of GVHD was compared among these groups by clinical and histologic grading. Twenty- eight of 28 mice treated with TBI + D0 SP died of acute GVHD, with overwhelming diarrhea by day 15 posttransplantation. Sixteen mice treated with either TBI + D4 SP or TBI + D7 SP developed acute GVHD, but none of them died of this disorder during 30 days posttransplantation. The mice conditioned with non-TBI regimens developed chronic GVHD within 3 months without showing any detectable signs of acute GVHD. Serum and in situ colonic cytokines were determined by enzyme-linked immunosorbent assay and immunohistology respectively. TBI itself significantly increased both serum and colonic tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and IL-6 when compared with non-TBI regimens and normal controls. TNF-alpha appeared in the serum and colon 4 hours post-TBI and peaked in 24 hours, followed by increasing IL-1 alpha and then IL-6 levels. TNF-alpha and IL-1 alpha decreased rapidly within 3 to 5 days post-TBI if no allogeneic cells were transplanted. Histoincompatible transplantation augmented cytokine release, which remained elevated on day 10 in these animals. Mice treated with TBI + D0 SP developed the most severe acute GVHD and had the highest levels of TNF-alpha, IL-1 alpha, and IL-6. The BuCy2-conditioned mice had the lowest cytokine levels and developed no acute GVHD. When the mice transplanted with TBI + D0 SP were treated immediately with recombinant soluble human TNF receptor (rhuTNFR:Fc) 100 micrograms/d intraperitoneally and for the subsequent 15 days acute GVHD mortality was significantly reduced from 100% to 50% (P < .001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Severe Ulcerative Colitis, Dural Sinus Thrombosis, and the Lupus Anticoagulant

Thrombnembolic disease is a well-recognized but very uncommon complication of inflammatory bowel disease. The mechanisms of the increased risk of thrombosis are not well understood: although several coagulation abnormalities have been described in inflammatory bowel disease patients, it is not clear whether they actually contribute to hypercoagulation or whether they are nonspecific markers of inflammation. Antiphospholipid antibodies (anticardiolipin antibodies and/or lupus anticoagulant) have recently been associated with an increased risk of thrombosis, particularly cerebrovascular disease in young patients. We report the case of a 33-yr-old female with severe ulcerative colitis at first attack who developed thrombosis of the superior and inferior longitudinal dural sinuses. No risk factors for thrombosis or coagulation abnormalities were observed; however, lupus anticoagulant was detected in the serum. The patient was successfully treated with osmotic agents, prophylactic anticonvulsant, and antiplatelet therapy, combined with i.v. steroids. After 6 months, the colitis is in remission, and the neurological recovery is good even if not yet complete.     

Gastric emptying in infants with gastroesophageal reflux. Ultrasound evaluation before and after cisapride administration.

The present study aimed to evaluate gastric emptying in children with gastroesophageal reflux (GER) by means of real-time ultrasonography, on the basis of measurements of the cross-sectional area of the gastric antrum. Twelve children with GER were studied (seven males, five females; age range, 3-13 months) and compared with 12 normal control children (six males, six females; age range, 3-13 months). The diagnosis of GER was confirmed by 24-h esophageal pH-monitoring. The GER patients had a significantly greater antral area than the controls at 90, 105, and 120 min after eating a standard meal (cow's milk formula, 300 ml/m2 body surface area); in addition, final gastric emptying time was significantly greater in the patients than in the controls (145 +/- 36.9 versus 78.7 +/- 19.3 min; p less than 0.0025). After 8 weeks of treatment with cisapride (0.3 ml/kg, three times a day) 24-h esophageal pH-monitoring and ultrasonography studies were repeated in the patients. The total percentage reflux time was significantly lower (p less than 0.038), and ultrasonography showed a decreased antral area at all the various study times, with no significant difference between patients and controls; final gastric emptying time was also significantly lower than before treatment (p less than 0.009). Furthermore, in the GER patients there was a significant correlation between gastric emptying time and the sum of the various reflux times recorded in the 2 h after all meals over the 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute choke zone effects: Lessons from radioactive and fluorescent microspheres in a pig model muscle flap

BACKGROUND:

Choke vessels dilate and contract to regulate blood flow between adjacent arterial angiosomes. In skin flap surgery, when arterial inflow to an angiosome is ligated, choke vessels allow blood supply from an adjacent angiosome. In muscle flap surgery, the vascular anatomy is analogous to skin flaps; however, while it is established that the choke vessels will fully dilate irreversibly after two to three days, no study has yet analyzed the acute changes in each vascular region immediately following ligation of one pedicle.

OBJECTIVE:

To establish whether the choke vessels open or close immediately following ligation of a pedicle, and how this change affects blood flow in the adjacent proximal and distal vascular regions.

METHODS:

Radioactive and fluorescent microspheres in a pig model were used to study the regional intramuscular blood flow in each anatomical zone of a rectus abdominis flap. Blood flow measurements for each zone were calculated relative to the entire muscle at preligation, ligation and various times (15 min to 90 min) postligation.

RESULTS:

There was no statistically significant difference in blood flow across choke zones as a result of ligation. This signifies that the choke vessels do not significantly dilate to produce a statistically significant measureable change in blood flow.

CONCLUSIONS:

Given these results and previous literature findings, the anatomical presence of choke vessels in a muscle is the strongest determining factor for acute flap viability in surgery.