Randomized controlled trial of pilocarpine hydrochloride for the moderation of oral mucositis during autologous blood stem cell transplantation.

Pilocarpine hydrochloride has been reported to increase salivation and decrease oral mucositis in patients receiving head and neck radiotherapy, but there is only one report of its use in a cancer chemotherapy patient population. This prospective, double-blinded, randomized, placebo-controlled trial was undertaken to determine the efficacy of pilocarpine for the moderation of oral mucositis during autologous blood stem cell transplantation. Subjects were randomized to receive a 5 mg tablet of pilocarpine, or a placebo, during and following chemotherapy. Subjects were seen every other day and evaluated for gingival, oral, and oropharyngeal mucositis; nutrition; oral hygiene; eating; speaking; sleeping; pain at rest and/or with swallowing; and mouth dryness. We recorded the mean and highest scores and duration of problems, along with white blood cell counts and differentials, and the use of systemic narcotics for oral mucosal pain. We enrolled and randomized 36 subjects, and there were no statistically or clinically significant differences for the primary outcome of severity of mucositis and no clinically significant differences in any of the other outcome measures. Pilocarpine has no benefit for the moderation of the incidence, severity, or duration of mucositis in patients receiving autologous blood stem cell transplantation.

Treatment of chronic myelogenous leukemia with autologous bone marrow transplantation followed by roquinimex.

Unmanipulated autologous bone marrow transplant (ABMT) offers patients with chronic myelogenous leukemia (CML) a long-term survival of 10%, at best. Immunotherapy has a role in the myeloid leukemias, and there is increasing evidence that of all hematopoietic neoplasms, CML may be the most susceptible to immune regulation. Roquinimex is known to enhance T cell, NK cell and macrophage activity. A phase II study was initiated in March 1992 to evaluate the role of roquinimex in Ph chromosome-positive CML post ABMT. Patients were conditioned with busulfan/ cyclophosphamide followed by reinfusion of unmanipulated Ph-positive bone marrow stem cells (>1 x 108 NBC/kg). When engraftment of neutrophils (ANC) reached 100/microl, patients received oral roquinimex twice weekly, escalating to a maximal dose of 0.2 mg/kg in 2 weeks. Seventeen patients have entered the study; 11 in first chronic phase (CP1); two in second chronic phase (CP2) and four in accelerated phase (AP). All required significant myelosuppressive therapy prior to ABMT to maintain stable blood counts and most had also received prior interferon therapy. All patients survived the transplant. Subsequent toxicity consisted mainly of musculoskeletal aches and peripheral edema. Additionally, specific skin changes were observed including graft-versus-host-like disease and eccrine sweat gland necrosis. Eight out of 17 patients are alive 28-60 months post ABMT. Of the nine patients who died, two were in CP2 and three in AP. All patients in CP1 went into a complete hematological remission post ABMT and seven of the 11 patients had at least a major cytogenetic response (greater than 65% Ph-negative metaphases) at 1 year or beyond and four of the 11 patients had a complete cytogenetic response at 2 years or beyond. Cytogenetic response post transplant often developed over time and did not simply represent post ABMT engraftment with Ph-negative cells. The clinical and cytogenetic data in these patients are encouraging and suggest that roquinimex may have significant activity when given post ABMT to patients with Ph-positive CML.

Analysis of factors that correlate with mucositis in recipients of autologous and allogeneic stem-cell transplants.

PURPOSE: To identify predictors of oral mucositis and gastrointestinal toxicity after high-dose therapy. PATIENTS AND METHODS: Mucositis and gastrointestinal toxicity were prospectively evaluated in 202 recipients of high-dose therapy and autologous or allogeneic stem-cell rescue. Of 10 outcome variables, three were selected as end points: the peak value for the University of Nebraska Oral Assessment Score (MUCPEAK), the duration of parenteral nutritional support, and the peak daily output of diarrhea. Potential covariates included patient age, sex, diagnosis, treatment protocol, transplantation type, stem-cell source, and rate of neutrophil recovery. The three selected end points were also examined for correlation with blood infections and transplant-related mortality. RESULTS: A diagnosis of leukemia, use of total body irradiation, allogeneic transplantation, and delayed neutrophil recovery were associated with increased oral mucositis and longer parenteral nutritional support. No factors were associated with diarrhea. Also, moderate to severe oral mucositis (MUCPEAK > or = 18 on a scale of 8 to 24) was correlated with blood infections and transplant-related mortality: 60% of patients with MUCPEAK > or = 18 had positive blood cultures versus 30% of patients with MUCPEAK less than 18 (P =.001); 24% of patients with MUCPEAK > or = 8 died during the transplantation procedure versus 4% of patients with MUCPEAK less than 18 (P =.001). CONCLUSION: Gastrointestinal toxicity is a major cause of transplant-related morbidity and mortality, emphasizing the need for corrective strategies. The peak oral mucositis score and the duration of parenteral nutritional support are useful indices of gastrointestinal toxicity because these end points are correlated with clinically significant events, including blood infections and treatment-related mortality.

Autotransplantation for relapsed or refractory Hodgkin's disease: long-term follow-up and analysis of prognostic factors.

Seventy consecutive patients with refractory or relapsed Hodgkin's disease who received high-dose chemotherapy followed by autologous stem cell rescue were analyzed to identify clinically relevant predictors of long-term event-free survival. High-dose therapy consisted primarily of carmustine (BCNU), etoposide, cytarabine and cyclophosphamide (BEAC). The 5-year Kaplan-Meier event-free survival (EFS) for the entire cohort was 32% (95% confidence interval; 18-45%) with a median follow-up of 3.6 years (range 7 months-7.6 years). The most significant predictor of improved survival was the presence of minimal disease (defined as all areas < or =2 cm) at the time of transplant: the 5 years EFS was 46 vs 10% for patients with bulky disease (P = 0.0002). Other independent predictors identified by step-wise regression analysis included the presence of non-refractory disease and the administration of post-transplant involved-field radiotherapy (XRT). Treatment-related mortality occurred in 13 of 70 patients: nine patients (13%) died within the first 100 days, mainly from cardiopulmonary toxicity. However, only one of 24 patients (4%) transplanted during the last 4.5 years died from early treatment-related complications. While high-dose therapy followed by autotransplantation led to long-term EFS of 50% for patients with favorable prognostic factors, a substantial proportion of patients relapsed, indicating that new therapeutic strategies are needed.

Treatment of multifocal lymphoma of bone with intensified ProMACE-CytaBOM chemotherapy and involved field radiotherapy.

Primary bone involvement is an unusual extranodal presentation of non-Hodgkin lymphoma (NHL). The optimal treatment for this entity has not been determined. While solitary bone lymphomas can be eradicated with local radiation in 50% of patients, distant relapses occur frequently, and the treatment of patients with multifocal osseous disease, or those presenting with associated soft tissue invasion or adenopathy is even less satisfactory. Over a 4-year period, nine patients with multifocal bone lymphoma were treated with intensified versions of the ProMACE-CytaBOM regimen and involved-field radiation. Seven patients had diffuse large cell histology and two patients had diffuse mixed type. Seven patients survived event-free at a median follow-up of 2.3 years (range .5-3.5). In most survivors, there was little or no change in the abnormal radiographic bone findings despite the clinical response to therapy. In one patient, magnetic resonance imaging (MRI) established that bone infarction rather than relapse of lymphoma was the cause of a new lytic bone lesion that developed during treatment.

Pathogenesis and management of paroxysmal nocturnal haemoglobinuria.

Over the past 30 years, our understanding of the pathogenesis of paroxysmal nocturnal haemoglobinuria (PNH) has increased dramatically. During that time, the events during complement activation and regulation have been described, the molecular basis for the exaggerated complement sensitivity of PNH cells has been uncovered, and the responsible gene mutation has been identified. It is now possible to relate almost all the protean manifestations of PNH to a single gene mutation in a haematopoietic stem cell. Unfortunately, our ability to manage these patients has not kept pace, and, with the exception of bone marrow transplantation, our major efforts are still directed toward control of complications rather than interruption of the disease process.

Autotransplantation for relapsed or refractory non-Hodgkin's lymphoma (NHL): long-term follow-up and analysis of prognostic factors.

One hundred and thirty-six patients autografted for relapsed or refractory non-Hodgkin's lymphoma (NHL) were evaluated to assess long-term event-free survival and to identify important prognostic factors. High-dose therapy consisted primarily of carmustine (BCNU), etoposide, cytarabine, and cyclophosphamide (BEAC) followed by unpurged autologous stem cell rescue. The 5-year Kaplan-Meier event-free survival (EFS) for the entire cohort was 34% (95% confidence interval: 24-44%) with a median follow-up of approximately 3 years (range 0-7.5 years). For patients entering with minimal disease (defined as all areas < or = 2 cm), the 5-year EFS was 40 vs 26% for those entering with bulky disease (P = 0.0004). In the multivariate analysis, minimal disease on entry and administration of involved-field XRT post-transplant were significantly associated with improved EFS; the latter association was observed mainly in the cohort of patients with bulky disease. The overall 100-day treatment-related mortality rate was 4.4% (3% for the last 71 patients). New strategies are needed to reduce the high rate of relapse (50-60%) following auto-transplantation for relapsed or refractory NHL.

Treatment of the neutropenia of Felty syndrome.

This review sets out to synthesize and critically evaluate the current reported data regarding therapeutic options for the neutropenia associated with Felty syndrome (Felty neutropenia). A MEDLINE search and bibliographies from recent reviews were used to identify trials and case reports that provided sufficient data to evaluate the effect of various interventions on both the neutropenia and the clinical course of patients with Felty syndrome. Data were obtained on baseline hematologic profiles, bone-marrow biopsies, and patient characteristics; length of follow-up; hematologic and clinical responses to the various interventions; and side-effect profiles. Treatment with hemopoietic growth factors or methotrexate can produce sustained hematologic and clinical responses with an acceptable side-effect profile. Splenectomy produces a long-term hematologic response in 80% of patients. Patients who do not respond hematologically have a higher incidence of non-fatal infections, but a significant minority (46%) do not experience any infections; the incidence of fatal infections is 12%, regardless of whether a hematologic response occurs. Of the patients who had infections prior to surgery, 55% did not experience further infections after splenectomy. Initial treatment of Felty neutropenia should consist of hemopoietic growth factors because of their rapid onset of action and relatively low incidence of side-effects. Splenectomy is a reasonable option if growth factors are ineffective and rapid amelioration of neutropenia is needed. Methotrexate offers a potentially promising alternative for the treatment of both the rheumatologic and the hematologic manifestations of Felty syndrome.

Changes in cytoskeletal actin content, F-actin distribution, and surface morphology during HL-60 cell volume regulation.

Cell volume regulation occurs via the regulated fluxes of ions and solutes across the cell membrane in response to cell volume perturbations under anisotonic conditions. Our earlier studies in human promyelocytic leukemic HL-60 cells showed that volume-dependent changes in total cellular F-actin content occur concomitantly as an inverse function of acute cell volume changes in anisotonic media (Hallows et al., 1991, Am. J. Physiol., 261:C1154-C1161). Although treatment with cytochalasin under anisotonic conditions significantly reduced total cellular F-actin levels, cytochalasin did not significantly affect the ability of cells to undergo normal volume regulation responses, which suggested that these volume-dependent changes in F-actin content may not play a critical role in HL-60 cell volume regulation. To examine more closely the possible role of the actin cytoskeleton in HL-60 cell volume regulation, we quantitated changes in Triton-insoluble cytoskeletal actin in the presence and absence of cytochalasin and also observed changes in F-actin distribution and surface morphology during volume regulation. The quantity of cytoskeletal-associated F-actin, like total F-actin, shifts inversely with initial cell volume changes in anisotonic media; however, subsequent changes in cytoskeletal actin levels during volume regulation are not significant. The soluble F-actin pool in HL-60 cells may thus be more susceptible to the physicochemical effects of shifts in cell volume than the insoluble (cytoskeletal) F-actin pool. Twenty-five micromolar dihydrocytochalasin B (DHB) treatment dramatically lowers cellular cytoskeletal actin levels by approximately 75% under resting (isotonic) conditions, but there are no significant further changes in cytoskeletal actin as cells undergo anisotonic volume regulation in the presence of DHB. These results suggest that volume-dependent changes in the absolute amounts of cytoskeletal-associated F-actin are not critical for HL-60 cell volume regulation. However, because some portions of the actin cytoskeleton are resistant to cytochalasin disruption during volume regulation, a role for the cytoskeleton in the sensing and signaling of HL-60 cell volume regulatory responses cannot be rigorously excluded. Particular F-actin distribution patterns, as observed using confocal fluorescent microscopy, were correlated with particular phases of volume regulation. Also, comparison of cellular F-actin distribution with surface morphology (observed by scanning electronic microscopy) of cells during volume regulation reveals a positive correlation between surface blebs and increased cortical F-actin staining intensity.

The effect of dexamethasone on functional properties of HL60 cells during all-trans retinoic acid induced differentiation. Are there implications for the retinoic acid syndrome?

Differentiation therapy for acute promyelocytic leukemia (APL) using all-trans-retinoic acid (ATRA) has improved the prognosis of the disease. ATRA therapy also causes a newly recognized clinical syndrome, the "retinoic acid syndrome" (RAS), which can be successfully managed with dexamethasone. Because aberrant function of maturing leukemic granulocytes may cause this syndrome, and because dexamethasone is useful clinically, we studied functional properties of maturing HL60 cells cultured in the presence and absence of dexamethasone. HL60 cells were cultured for 4 days with ATRA and studied daily to determine acquisition of mature neutrophil-like properties including phagocytosis, NBT reduction, actin polymerization, chemotaxis and adhesion molecule expression. Undifferentiated HL60 cells could not polymerize actin or reduce NBT, and exhibited only a minimal ability to undergo chemotaxis or ingest latex beads. Following 4 days of maturation with ATRA, the cells would increase F-actin content in response to interleukin-8, ingest latex beads, migrate in a chemotaxis chamber, reduce NBT, and express CD11b. When dexamethasone was added to the cells in culture, there was no major enhancement or suppression of these properties. We also studied the effect of dexamethasone on functional properties of normal neutrophils and found minimal if any effect on their function. Overall, these studies suggest that in vitro, dexamethasone has little effect on the function of leukemic and normal granulocytes. To further investigate the pathophysiology of the retinoic acid syndrome, future studies may need to use endothelial cells, cytokines, or granulocytes obtained from APL patients.

Functional characteristics of mature granulocytes in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid.

All-trans retinoic acid (ATRA) is a differentiating agent that has been successfully used in the treatment of patients with acute promyelocytic leukemia (APL). Functional properties of peripheral blood neutrophils from a patient with APL during treatment with ATRA have been studied. Wright stain of patient neutrophils showed hypogranulation and loose nuclear chromatin when compared with normal neutrophils. These cells were of lower density than normal neutrophils and separated on density gradient centrifugation with mononuclear cells. Surface antigen expression by FACS distinguished these cells from lymphocytes. The histograms showed a population of larger cells expressing CD18 and CD11b, distinct from the smaller cells which did not express CD11b. fMLP caused an increase in intracellular calcium (measured spectrophotometrically) that was inhibited by the calcium chelator BAPTA. Actin polymerization following cell activation was measured using NBD-phallacidin staining and FACS. Both IL-8 and fMLP caused rapid increases using F-actin content (2.5-3.0 fold), which were of greater magnitude than generally seen with normal neutrophils. Treatment with BAPTA before activation with fMLP did not blunt the actin responses, despite complete inhibition of an intracellular calcium increase. In summary, neutrophils derives from differentiated APL cells express CD18/CD11b, and exhibit a similar degree of actin polymerization in response to fMLP and IL-8, independent of an increase in intracellular calcium. Although the actin responses are greater than normal neutrophils, most properties are similar, supporting the contention that these cells can protect the host. The exaggerated actin response to inflammatory mediators, however, may play a role in the 'retinoic acid syndrome'.

Patients > or = age 40 years undergoing autologous or allogeneic BMT have regimen-related mortality rates and event-free survivals comparable to patients < age 40 years.

To evaluate the safety and efficacy of marrow transplantation for older adults, the regimen-related mortality and event-free survival for patients > or = 40 years were compared with those for patients < 40 years. Of 148 consecutive patients receiving autotransplants for lymphoma or Hodgkin's disease, 70 were < 40 years and 78 were > or = 40 years at the time of transplant, including 40 who were > or = 50 years and 12 who were > or = 60 years. Eleven patients (16%) in the younger age group died from transplant-related complications compared with 4 (5%) in the older age group. The 4-year actuarial event-free survivals (EFS) for the younger and older age groups were 43% and 48%, respectively. After adjustment for covariates with prognostic significance, older age was marginally associated with improved event-free survival (P = 0.08). Of 92 consecutive patients undergoing allogeneic BMT during the same period, 62 patients were < 40 years and 30 patients were > or = 40 years, including 8 patients > or = 50 years, and 1 patient > 60 years. Non-relapse mortality (including deaths from GVHD) occurred in 28 of the younger patients (45%) and 9 of the older patients (30%). The 3-year actuarial EFS for the younger patients was 26% vs. 56% for the patients > or = 40 years (P = 0.057). However, this difference was mainly due to the higher proportion of patients with CML and early-stage leukemia in the older age group.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional properties of HL60 cells matured with all-trans-retinoic acid and DMSO: differences in response to interleukin-8 and fMLP.

All-trans-retinoic acid (ATRA) causes granulocyte differentiation in patients with acute promyelocytic leukemia. HL60 cells are frequently used as an in vitro model for studying granulocytes during maturation. We have previously studied actin polymerization in response to fMLP in HL60 cells undergoing DMSO induced maturation, and reported that IL-8 causes actin polymerization in neutrophils in a manner similar to fMLP. We now compare chemotaxis and actin polymerization in response to IL-8 and fMLP, and nitroblue tetrazolium (NBT) reduction in HL60 cells matured with ATRA and DMSO. Cells cultured for 4 days with ATRA and DMSO showed morphologic evidence of maturation. NBD-phallacidin staining and flow cytometry were used to measure changes in F-actin content in response to IL-8 and fMLP. Uninduced cells were not capable of actin polymerization or chemotaxis. Cells matured with ATRA exhibited a 2.6-fold increase in F-actin content in response to IL-8, but only a 1.2-fold increase in response to fMLP. Cells matured with DMSO responded to both IL-8 and fMLP in an equal manner with 1.6-fold increases in F-actin. The 2 h migration for ATRA induced cells was 124 microns in response to IL-8, 107 microns with fMLP, and 105 microns in buffer. DMSO induced cells migrated 89 microns in response to IL-8, 106 microns with fMLP, and 66 microns in buffer. With maturation, 65% of the ATRA induced cells reduced NBT compared with only 15% of the DMSO induced cells. In summary, HL60 cells cultured in ATRA develop greater functional maturity than those cultured in DMSO, and a greater responsiveness to IL-8 than fMLP, a finding distinct from previously reported work in neutrophils.

The Rochester practice-based experience. An experiment in medical education.

BACKGROUND: Recent changes in the organization and financing of medical practice have resulted in a shift of patient care from the hospital to ambulatory locations and dramatically changed the mix and severity of the inpatient population. Medical educators are concerned that hospitalized patients are not truly representative of the practice of internal medicine and may be unsuitable for the education of third-year medical students. The University of Rochester (NY) Department of Medicine recently established a "practice-based" component to complement the inpatient segment of the third-year medicine clerkship. METHODS: Participating students work with a practicing internist for the second half of the 12-week clerkship. Students evaluate office and hospital patients in the internist's practice and attend didactic conferences. We used a multi-measurement curriculum evaluation system to assess the new program and compare it with the established hospital experience. Evaluation components included the following: logs in which students profiled their clinical activities; questionnaires that assessed opinions and satisfaction about the clerkship; group interviews with students; and visits to practice-based sites to interview both preceptor and student. RESULTS: Practice-based students saw many more patients with a broader range of illnesses and had more observation and supervision from faculty than hospital-based students. Practice-based students discussed all of their patients with their preceptors, who were perceived as being excellent teachers. Hospital-based students, although generally positive, were also generally less enthusiastic about the amount and quality of teaching, observation, and supervision. Practice-based and hospital-based students achieved similar grades on the final examination and received a similar number of honors grades. Preceptors were enthusiastic in their endorsement of the practice-based experience and patient acceptance of students was high. CONCLUSION: The practice-based experience is feasible and of equal academic rigor to the hospital experience. This realistic immersion into the daily activities of an internist provides an enriching balance to the traditional hospital-based clerkship.

Engagement of the T-cell antigen receptor by anti-CD3 monoclonal antibody causes a rapid increase in lymphocyte F-actin.

Activation of protein kinase C (PKC) causes a rapid and sustained increase in the F-actin of T lymphocytes. Because the phosphatidylinositol pathway and the cytoskeleton play a role in lymphocyte activation, we examined the relationship between signal transduction and the F-actin increase in human blood T cells. Anti-CD3 monoclonal antibodies (mAbs) initiate signals which result in activation of T lymphocytes through the T-cell receptor (TCR), involving the phosphatidylinositol pathway, activation of PKC, and increasing intracellular calcium (Cai2+). The fluorescent probe NBD-phallacidin was used to examine the conformational state of actin following stimulation of T lymphocytes with anti-CD3 mAb. Each of three different murine anti-CD3 mAbs caused rapid increases in lymphocytic F-actin content, which was enhanced by cross-linking with a goat anti-mouse IgG. A maximally effective dose of the mAb Leu 4 caused a rise in cellular F-actin of 1.8-fold at 2 minutes and a three-fold increase in Cai2+. Ionomycin, 100 nM, caused a Cai2+ rise similar in magnitude to that caused by anti-CD3 mAb but had no effect on F-actin content. Inhibitors of PKC, 1(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), sphingosine, and sphinganine lowered the resting cellular F-actin and partially blocked the increase in F-actin caused by either anti-CD3 mAb or ionomycin; however, they had no effect on the rise in Cai2+. Cells leached of Ca2+ with EGTA and ionomycin exhibited no Cai2+ increase in response to anti-CD3 mAb or ionomycin; such cells retained the F-actin increase caused by anti-CD3 mAb. We conclude that stimulation of human T lymphocytes via the TCR causes an early rapid increase in F-actin content. Activation of PKC may play a role but the concomitant Cai2+ increase is neither sufficient nor necessary for the F-actin increase.

One hundred autotransplants for relapsed or refractory Hodgkin's disease and lymphoma: value of pretransplant disease status for predicting outcome.

PURPOSE: One hundred autotransplants for Hodgkin's disease (HD) or non-Hodgkin's lymphoma (NHL) were examined prospectively to identify variables with prognostic significance. PATIENTS AND METHODS: Ninety-six patients with relapsed or refractory HD or NHL underwent 100 autotransplants. Patients received high-dose carmustine (BCNU), etoposide, cytarabine, and cyclophosphamide (BEAC) followed by unpurged autologous stem-cell rescue. RESULTS: The 3-year actuarial event-free survival (EFS) rate for the 47 HD patients is 49%, with a median followup duration of 2 years. For the 53 NHL patients, the 3-year actuarial EFS rate is 40%, with a median follow-up duration of 19 months. By multivariate analysis, minimal disease on admission (all areas < or = 2 cm) is associated with improved EFS (HD, P = .003, NHL, P = .03). The projected EFS rate for HD patients entering with minimal disease is 70% versus 15% for patients with bulky disease (P = .0001). The projected EFS rate for NHL patients with minimal disease is 48% versus 25% for patients with bulky disease (P = .04). Posttransplant involved-field radiotherapy, administered to 26 of the last 61 patients, was associated with an improved EFS rate for NHL patients (P = .015). The BEAC regimen was well tolerated by patients who entered the study with minimal disease (mortality rate, < 5%), but caused significant toxicity in patients with bulky disease (mortality rate, 25%). CONCLUSION: Disease burden before autotransplantation is an important predictor of regimen-related toxicity and EFS. Posttransplant involved-field radiotherapy may improve outcomes in select patients with NHL. The BEAC regimen is safe and effective, particularly for patients with minimal disease.

The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified.

Tumor cells expressing the herpes simplex virus thymidine kinase (HSV-TK) gene are sensitive to the drug ganciclovir (GCV). We demonstrate here that HSV-TK-positive cells exposed to GCV were lethal to HSV-TK-negative cells as a result of a "bystander effect." HSV-TK-negative cells were killed in vitro when the population of cultured cells contained only 10% HSV-TK-positive cells. The mechanism of this "bystander effect" on HSV-TK-negative cells appeared to be related to the process of apoptotic cell death when HSV-TK-positive cells were exposed to GCV. Flow cytometric and electron microscopic analyses suggested that apoptotic vesicles generated from the dying gene-modified cells were phagocytized by nearby, unmodified tumor cells. Prevention of apoptotic vesicle transfer prevented the bystander effect. The toxic effect of HSV-TK-positive cells on HSV-TK-negative cells was reproduced in an in vivo model. A mixed population of tumor cells consisting of HSV-TK-positive and HSV-TK-negative cells was inoculated s.c. into mice. Regression of the tumor mass occurred when the inoculum consisted of as few as 10% HSV-TK-expressing tumor cells. The bystander effect was also demonstrated in i.p. tumor studies. Initial experiments demonstrated that prolonged survival (> 70 days) occurred when a mixture containing 50% HSV-TK-positive and 50% HSV-TK-negative cells was injected i.p. followed by GCV treatment. Further, survival was prolonged for mice with a preexisting HSV-TK-negative i.p. tumor burden by injecting HSV-TK-positive cells and GCV. These results suggest that genetic modification of tumor cells may be useful for developing cancer therapies.

Signal pathway regulation of interleukin-8-induced actin polymerization in neutrophils.

Interleukin-8 (IL-8), a recently described peptide cytokine, is a neutrophil chemoattractant and activator that exerts effects similar to fMLP, yet their receptors and their roles in pathophysiology differ. The effect of IL-8 on the neutrophil cytoskeleton has not been well studied; therefore, we compared and contrasted the effects of IL-8 and fMLP on neutrophil actin conformation and on the signal pathway regulation of actin responses. IL-8 caused a rapid, dose-dependent increase in neutrophil F-actin content within 30 seconds. The maximum increase was twofold. These changes were accompanied by the development of F-actin-rich pseudopods, as noted with fluorescence microscopy and scanning electron microscopy. Selected biochemical inhibitors were used to study the regulation of the IL-8-induced actin changes. Incubation of neutrophils with 2 micrograms/mL pertussis toxin resulted in a 67% inhibition of the IL-8-induced F-actin increase. The protein kinase C (PKC) inhibitors, staurosporine and H7, did not inhibit the increase in F-actin caused by IL-8. IL-8 caused a rapid increase in neutrophil intracellular calcium that could be completely inhibited by the chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid (BAPTA). However, BAPTA-treated neutrophils retained the ability to increase F-actin in response to IL-8. Similar results were seen with fMLP, indicating that, similar to fMLP, the IL-8-induced actin response is mediated through pertussis-toxin-sensitive G-proteins but is neither dependent on PKC nor increases in cytosolic calcium. Thus, although IL-8 and fMLP exert their effects on neutrophils through different receptors, the signal transduction pathways used and the effects on actin conformation and pseudopod formation are similar.

Acute cell volume changes in anisotonic media affect F-actin content of HL-60 cells.

To investigate the possible role of the cytoskeleton in volume regulatory responses of human promyelocytic leukemic (HL-60) cells, we monitored and modulated the F-actin content of these cells undergoing volume regulation in anisotonic media. Initial volume changes of HL-60 cells suspended in hypertonic media followed a Van't Hoff relationship, and intracellular F-actin content during volume regulatory responses in anisotonic media changed concomitantly as an inverse function of the volume shifts. These F-actin changes were shown to be an explicit function of cell volume and not tonicity of the medium. The data fit with the idea that changes in affinity of actin-binding proteins (ABPs) for actin and/or changes in the overall effective critical concentration of actin occur during acute cell volume changes, producing shifts in the relative amounts of G- and F-actin. Treatment of HL-60 cells with dihydrocytochalasin B (DHB), which perturbs cellular actin assembly, lowered resting levels of intracellular F-actin but did not prevent volume-associated F-actin changes in anisotonic media. Despite the lowered F-actin levels, HL-60 cells in the presence of DHB still undergo normal volume regulatory responses. Thus the absolute amount of intracellular F-actin does not appear to be critical for volume regulation in HL-60 cells.