Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maternal dietary ethanol consumption is associated with hypertriglyceridemia in adult rat offspring

BACKGROUND: The consumption of significant amounts of alcohol (ethanol, EtOH) may markedly increase serum triglyceride levels. This study describes a significant increase in fasting serum triglyceride (TG) levels in adult male rats whose mothers consumed EtOH. The hypertriglyceridemia occurred although the offspring never directly consumed EtOH and had consumed only rat chow for the preceding 14 months. Furthermore, both male and female adult offspring had an additional, significant increase in TG levels if their mothers consumed EtOH and experienced stress (restraint) during the pregnancy. METHODS: Harlan-derived Sprague Dawley female rats were dosed during pregnancy with EtOH via a liquid diet, and their offspring were compared with offspring of mothers who were either fed ad libitum or pair-fed the liquid diet without EtOH. At birth, the offspring of EtOH mothers exhibited no visible abnormalities except reduced weight, and all offspring were surrogate fostered within 48 hr of birth to mothers who had consumed commercial rat chow throughout their pregnancy. After weaning, all offspring consumed only commercial rat chow, and they were examined over the next 14 months for changes in triglyceride homeostasis as a function of maternal alcohol intake. RESULTS: Adult male offspring of mothers that consumed EtOH during their pregnancy had significant increases in fasting serum triglycerides associated with an increase in the very low density lipoprotein serum fraction. Acute administration of insulin to the offspring of all maternal dietary groups resulted in a rapid clearing of the serum triglycerides, and there were no differences in basal or heparin-releasable lipoprotein lipase activity between any of the progeny. Castration of the male offspring of EtOH-treated mothers prevented the development of elevated TG levels. Administration of testosterone to littermate female offspring increased circulating TG levels compared with testosterone-treated offspring of pair-fed mothers. EtOH-consuming mothers who also underwent five periods of restraint-induced stress (approximately 10 min each session) produced offspring whose fasting serum TG levels were higher than those whose mothers consumed EtOH but experienced no restraint or who experienced restraint but no EtOH. Maternal stress significantly reduced lipoprotein lipase activity in some offspring treatment groups, but the changes did not correspond to changes in the serum TG levels of the offspring. That is, maternal restraint-induced stress was associated with a loss of heparin-releasable lipoprotein lipase activity by male progeny from pair-fed and EtOH-fed mothers and the female offspring of ad libitum-fed and EtOH-fed mothers. CONCLUSIONS: Although serum triglycerides increased with age in all offspring, the increase was much more pronounced in the progeny of mothers who consumed EtOH during their pregnancy. The hypertriglyceridemia was significantly more pronounced in the male offspring and in female offspring treated with testosterone. Castration of male offspring inhibited the hypertriglyceridemia development, which suggests that male sex hormones may play a role in the development of this condition. Maternal EtOH consumption coupled with maternal restraint-induced stress significantly increased the level of hypertriglyceridemia in both male and female offspring compared with offspring whose mothers experienced restraint but no EtOH or EtOH with no restraint. If this study models the human condition, the results could represent an unrecognized risk factor in a number of adult disease states hypothesized to be associated with hypertriglyceridemia, such as cardiovascular disease, hypertension, and diabetes.     

Detection of anti-HTLV-I Tax antibodies in HTLV-I enzyme-linked immunosorbent assay-negative individuals

The HTLV-I tax gene protein (Tax) is not packaged within the mature viral particle from which the proteins for the commercially available enzyme-linked immunosorbent assay (ELISA) are derived. Screening of 162 individuals within a cohort of white intravenous (IV) drug abusers, previously identified as having an increased incidence of HTLV-I infection, demonstrated that seven of them had antibodies to the HTLV-I Tax protein but tested negative in HTLV-I ELISAs and Western blots prepared from purified virion proteins. Three out of 35 individuals in other behaviorally defined high-risk groups also displayed this limited pattern of reactivity to HTLV-I proteins. The presence of the anti-HTLV- I p40/Tax antibodies was determined by radioimmunoprecipitation assay (RIPA), which also revealed low levels of anti-env reactivity. The specificity of the anti-p40 reactivity was confirmed on specific Tax ELISAs and Western blots prepared from recombinantly produced Tax. In vitro gene amplification by the polymerase chain reaction (PCR) was used to establish the presence of sequences homologous to HTLV-I proviral DNA in four/four of these HTLV-I ELISA negative, Tax ELISA/Tax western blot/RIPA positive individuals. These data suggest that the true incidence of HTLV-I infection within high-risk cohorts is greater than previously reported.     

Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia

A phase I clinical trial was initiated to treat patients with stage IV B-derived chronic lymphocytic leukemia (CLL) with the IgG2a murine monoclonal antibody T101. This antibody binds to a 65,000-mol wt (T65) antigen found on normal T lymphocytes, malignant T lymphocytes, and B- derived CLL cells. All of the patients had a histologically confirmed diagnosis of advanced B-derived CLL and were refractory to standard therapy, and more than 50% of their leukemia cells reacted with the T101 antibody in vitro. The patients received T101 antibody two times per week, over two to 50 hours by intravenous administration in 100 mL of normal saline containing 5% human albumin. Twelve patients were treated with a fixed dosage of 1, 10, 50, or 100 mg, and one patient was treated with 140 mg of antibody. It was demonstrated that patients given two-hour infusions of 50 mg developed pulmonary toxicity, with shortness of breath and chest tightness. This toxicity was eliminated when infusions of 50 or 100 mg of T101 were prolonged to 50 hours. All dose levels caused a rapid but transient decrease in circulating leukemia cell counts. In vivo binding to circulating and bone marrow leukemia cells was demonstrated at all dose levels with increased binding at higher dosages. Antimurine antibody responses were not demonstrated in any patients at any time during treatment. Circulating free murine antibody was demonstrated in the serum of only the two patients treated with 100 mg of antibody as a 50-hour infusion and the patient treated with 140 mg of antibody over 30 hours. Antigenic modulation was demonstrated in patients treated at all dose levels but was particularly apparent in patients treated with prolonged infusions of 50 and 100 mg of antibody. We were also able to demonstrate antigenic modulation in lymph node cells, which strongly suggests in vivo labeling of these cells. Overall, T101 antibody alone appears to have a very limited therapeutic value for patients with CLL. The observations of in vivo labeling of tumor cells, antigenic modulation, antibody pharmacokinetics, toxicity, and antimurine antibody formation may be used in the future for more effective therapy when drugs or toxins are conjugated to the antibody.     

Murine CD27(?) Vγ6(+) γδ T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages

Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include γδ T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-γ. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, γδ T cells promote tumor cell growth. γδ T cells accumulated in the peritoneal cavity in response to tumor challenge and could be visualized within solid tumor foci. Functional characterization of tumor-associated γδ T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-γ. Consistent with this finding, both T cell receptor (TCR)δ-deficient and IL-17–deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by γδ T cells in the tumor environment was essentially restricted to a highly proliferative CD27⁽⁻⁾ subset that expressed Vγ6 instead of the more common Vγ1 and Vγ4 TCR chains. The preferential expansion of IL-17–secreting CD27⁽⁻⁾ Vγ6⁽⁺⁾ γδ T cells associated with the selective mobilization of unconventional small peritoneal macrophages (SPMs) that, in comparison with large peritoneal macrophages, were enriched for IL-17 receptor A, and for protumor and proangiogenic molecular mediators, which were up-regulated by IL-17. Importantly, SPMs were uniquely and directly capable of promoting ovarian cancer cell proliferation. Collectively, this work identifies an IL-17–dependent lymphoid/myeloid cross-talk involving γδ T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.Developing tumors are infiltrated by a variety of leukocyte subsets that can either promote or inhibit inflammation, and thus impact on cancer progression (1). Among such populations are γδ T cells, which are major players in lymphoid stress surveillance likely due to their recognition of stress-inducible molecules independently of MHC-mediated antigen presentation (2). Moreover, abundant IFN-γ secretion and cytotoxic effector functions endow γδ T cells with potent antitumor activity. This has been clearly documented in murine models of spontaneous (3), chemically induced (4), transgenic (5), and transplantable (6, 7) tumors. For example, in the widely used B16 melanoma model, γδ T cells were shown to infiltrate tumors very early and provided a critical source of IFN-γ that significantly delayed tumor growth (6, 7).Human γδ T cells also possess IFN-γ–secreting potential, which is displayed immediately at birth (8) and display cytotoxicity against tumor lines of diverse origin, including epithelial (9, 10) and hematological (11, 12) tumors. This has prompted the development of cancer clinical trials targeting γδ T cells, which have produced encouraging, albeit highly variable, degrees of therapeutic responses (13–15). There is therefore great interest in maximizing the antitumor functions of γδ T cells for cancer immunotherapy.Despite these highly promising reports, a clinical study on breast cancer tissue revealed a surprising inverse correlation between infiltrating γδ T cells and overall patient survival (16). In fact, γδ T cells represented the most significant independent prognostic factor for assessing severity of breast cancer (16). Similarly, a recent report on colorectal cancer showed a positive correlation between clinopathological parameters and the infiltration of γδ T cells specifically producing interleukin-17 (IL-17) (17). A tumor-promoting function of γδ T cells was also suggested in murine fibrosarcoma (18) and hepatocellular carcinoma (19) models, in which γδ T cells were the major cellular source of IL-17, which was required for optimal tumor growth in vivo. These data raise the interesting question as to whether distinct functional attributes of γδ T cells, for example differential cytokine production, may associate with markedly different outcomes for tumor growth.Along these lines, we have pioneered the identification of two distinct functional subsets of murine γδ T cells based on the expression levels of the CD27 coreceptor (20). We showed that robust IFN-γ production is associated with the CD27⁽⁺⁾ phenotype, whereas secretion of IL-17 is restricted to CD27⁽⁻⁾ γδ T cells. This dichotomy of hard-wired commitment to specific cytokine production is established during thymic development and maintained during the immune response to various infection agents (21, 22). Thus, the overall impact of γδ T cells in a given disease may depend on the balance between distinct proinflammatory effector cell subsets.Building on these foundations, we have here analyzed the overall and subset-specific contributions of γδ T cells to a well-established murine syngeneic model of ovarian cancer (ID8; transplantable cell line) that has a strong inflammatory component (23–25), akin to that observed in human patients with high-grade serous ovarian cancer (25, 26). In this murine model, we demonstrate that γδ T cells are major sources of IL-17, and both T cell receptor (TCR)δ-deficient and IL-17–deficient mice display reduced ID8 tumor growth. Interestingly, IL-17 production by γδ T cells in the tumor environment is essentially restricted to a CD27⁽⁻⁾ subset that does not express the commonly used Vγ1 or Vγ4 TCR chains, but rather Vγ6; these Vγ6⁽⁺⁾ cells are highly biased toward IL-17 production, in contrast to their IFN-γ–producing Vγ1⁽⁺⁾ and Vγ4⁽⁺⁾ counterparts. The ID8 tumor environment gets progressively enriched in the IL-17–promoting factor IL-7, whose receptor is highly expressed on Vγ6⁽⁺⁾ cells. This associates with preferential Vγ6⁽⁺⁾ cell proliferation and accumulation of IL-17 in the tumor bed, which in turn induces the mobilization of (recently described) small peritoneal macrophages (SPMs) that are enriched in IL-17 receptor A (IL-17RA) and in protumor and proangiogenic molecular mediators. Importantly, in comparison with large peritoneal macrophages (LPMs), SPMs can strongly and directly promote ovarian cancer cell proliferation. In summary, our work identifies an IL-17–dependent γδ T-cell/SPM axis that promotes tumor cell growth and thus opposes the widely accepted antitumor (and IFN-γ mediated) function of γδ T cells.     

Improving the Th1 cellular efficacy of the lead Yersinia pestis rF1-V subunit vaccine using SA-4-1BBL as a novel adjuvant

The lead candidate plague subunit vaccine is the recombinant fusion protein rF1-V adjuvanted with alum. While alum generates Th₂ regulated robust humoral responses, immune protection against Yersinia pestis has been shown to also involve Th₁ driven cellular responses. Therefore, the rF1-V-based subunit vaccine may benefit from an adjuvant system that generates a mixed Th₁ and humoral immune response. We herein assessed the efficacy of a novel SA-4-1BBL costimulatory molecule as a Th₁ adjuvant to improve cellular responses generated by the rF1-V vaccine. SA-4-1BBL as a single adjuvant had better efficacy than alum in generating CD4⁺ and CD8⁺ T cells producing TNFα and IFNγ, signature cytokines for Th₁ responses. The combination of SA-4-1BBL with alum further increased this Th₁ response as compared with the individual adjuvants. Analysis of the humoral response revealed that SA-4-1BBL as a single adjuvant did not generate a significant Ab response against rF1-V, and SA-4-1BBL in combination with alum did not improve Ab titers. However, the combined adjuvants significantly increased the ratio of Th₁ regulated IgG_2c in C57BL/6 mice to the Th₂ regulated IgG₁. Finally, a single vaccination with rF1-V adjuvanted with SA-4-1BBL + alum had better protective efficacy than vaccines containing individual adjuvants. Taken together, these results demonstrate that SA-4-1BBL improves the protective efficacy of the alum adjuvanted lead rF1-V subunit vaccine by generating a more balanced Th₁ cellular and humoral immune response. As such, this adjuvant platform may prove efficacious not only for the rF1-V vaccine but also against other infections that require both cellular and humoral immune responses for protection.     

The provision of a time-critical elective surgical service during the COVID-19 Crisis: a UK experience

IntroductionWith the emergence of the COVID-19 pandemic, all elective surgery was temporarily suspended in the UK, allowing for diversion of resource to manage the anticipated surge of critically unwell patients. Continuing to deliver time-critical surgical care is important to avoid excess morbidity and mortality from pathologies unrelated to COVID-19. We describe the implementation and short-term surgical outcomes from a system to deliver time-critical elective surgical care to patients during the COVID-19 pandemic.Materials and methodsA protocol for the prioritisation and safe delivery of time-critical surgery at a COVID-19 ‘clean’ site was implemented at the Nuffield Health Exeter Hospital, an independent sector hospital in the southwest of England. Outcomes to 30 days postoperatively were recorded, including unplanned admissions after daycase surgery, readmissions and complications, as well as the incidence of perioperative COVID-19 infection in patients and staff.ResultsA total of 128 surgical procedures were performed during a 31-day period by a range of specialties including breast, plastics, urology, gynaecology, vascular and cardiology. There was one unplanned admission and and two readmissions. Six complications were identified, and all were Clavien-Dindo grade 1 or 2. All 128 patients had preoperative COVID-19 swabs, one of which was positive and the patient had their surgery delayed. Ten patients were tested for COVID-19 postoperatively, with none testing positive.ConclusionThis study has demonstrated the implementation of a safe system for delivery of time-critical elective surgical care at a COVID-19 clean site. Other healthcare providers may benefit from implementation of similar methodology as hospitals plan to restart elective surgery.