Transmission potential of human schistosomes can be driven by resource competition among snail intermediate hosts.

Predicting and disrupting transmission of human parasites from wildlife hosts or vectors remains challenging because ecological interactions can influence their epidemiological traits. Human schistosomes, parasitic flatworms that cycle between freshwater snails and humans, typify this challenge. Human exposure risk, given water contact, is driven by the production of free-living cercariae by snail populations. Conventional epidemiological models and management focus on the density of infected snails under the assumption that all snails are equally infectious. However, individual-level experiments contradict this assumption, showing increased production of schistosome cercariae with greater access to food resources. We built bioenergetics theory to predict how resource competition among snails drives the temporal dynamics of transmission potential to humans and tested these predictions with experimental epidemics and demonstrated consistency with field observations. This resource-explicit approach predicted an intense pulse of transmission potential when snail populations grow from low densities, i.e., when per capita access to resources is greatest, due to the resource-dependence of cercarial production. The experiment confirmed this prediction, identifying a strong effect of infected host size and the biomass of competitors on per capita cercarial production. A field survey of 109 waterbodies also found that per capita cercarial production decreased as competitor biomass increased. Further quantification of snail densities, sizes, cercarial production, and resources in diverse transmission sites is needed to assess the epidemiological importance of resource competition and support snail-based disruption of schistosome transmission. More broadly, this work illustrates how resource competition can sever the correspondence between infectious host density and transmission potential.

Ten simple rules for training yourself in an emerging field.

The opportunity to participate in and contribute to emerging fields is increasingly prevalent in science. However, simply thinking about stepping outside of your academic silo can leave many students reeling from the uncertainty. Here, we describe 10 simple rules to successfully train yourself in an emerging field, based on our experience as students in the emerging field of ecological forecasting. Our advice begins with setting and revisiting specific goals to achieve your academic and career objectives and includes several useful rules for engaging with and contributing to an emerging field.

Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry.

Viral induction of autoimmunity is thought to occur by either bystander T-cell activation or molecular mimicry. Coxsackie B4 virus is strongly associated with the development of insulin-dependent diabetes mellitus in humans and shares sequence similarity with the islet autoantigen glutamic acid decarboxylase. We infected different strains of mice with Coxsackie B4 virus to discriminate between the two possible induction mechanisms, and found that mice with susceptible MHC alleles had no viral acceleration of diabetes, but mice with a T cell receptor transgene specific for a different islet autoantigen rapidly developed diabetes. These results show that diabetes induced by Coxsackie virus infection is a direct result of local infection leading to inflammation, tissue damage, and the release of sequestered islet antigen resulting in the re-stimulation of resting autoreactive T cells, further indicating that the islet antigen sensitization is an indirect consequence of the viral infection.

Entry of naive CD4 T cells into peripheral lymph nodes requires L-selectin.

Binding of L-selectin expressed on lymphocytes to carbohydrate ligand(s) on lymph node high endothelial venules is thought to initiate lymphocyte extravasation from blood to lymph during recirculation and localization to sites of antigen (Ag) exposure. Previous studies have shown that treatment of lymphocytes with antibody to L-selectin (MEL-14) ablates trafficking to peripheral lymph nodes (PLN). In mice, naive but not memory CD4 cells express L-selectin. To examine the role of L-selectin in helper T cell migration, we studied the effects of in vivo administration of MEL-14 on CD4 cell responses. Systemic exposure of mice to MEL-14 depleted CD4 cells expressing a naive phenotype (CD45RBhi, CD44lo) from PLN but not from spleen. The majority of residual lymph node CD4 cells exhibited the reciprocal, memory phenotype (CD45RBlo, CD44hi). MEL-14 treatment prevented priming of naive CD4 cells for proliferation and cytokine production (IL-2 and IL-4) to keyhole limpet hemocyanin in PLN draining the site of Ag injection, but not in the spleen. The results suggest that naive cells were not depleted, but rather diverted to other sites where priming occurred. The data demonstrate that L-selectin mediates extravasation of naive CD4 cells into PLN and that its function cannot be replaced by other homing receptors.

Characterization of antigen-specific CD4+ effector T cells in vivo: immunization results in a transient population of MEL-14-, CD45RB- helper cells that secretes interleukin 2 (IL-2), IL-3, IL-4, and interferon gamma.

In previous studies we demonstrated that, following activation by mitogens or alloantigens, helper T cell precursors proliferate and differentiate in vitro to produce a population of effector cells that secrete high titers of lymphokines upon restimulation. In this report, we demonstrate that a similar effector population develops in vivo following primary antigen stimulation. When restimulated with specific antigen in vitro, CD4+ T cells from mice primed 5 to 7 days previously by subcutaneous administration of keyhole limpet hemocyanin (KLH) in adjuvant, produced high levels of interleukin 2 (IL-2), IL-4, and IL-3, and little or no interferon gamma (IFN-gamma) or IL-5. The effector T cells provided excellent helper activity for in vitro antibody responses of 4-hydroxy-5-iodo-nitrophenyl acetic acid-primed B cells with the production principally of the immunoglobulin G1 (IgG1) and IgM isotypes, small quantities of IgG3, and no detectable IgG2a, or IgG2b. Antigen-specific secretion of IL-2, IL-3, and IL-4 by in vivo effectors was detectable by 12 hours following in vitro restimulation. IFN-gamma and IL-5 were not detected until 48 and 72 hours of culture, respectively, and low levels of these lymphokines were produced. Lymphokine production by primed CD4+ T cells could be induced as early as 3 days following immunization, peaked on day 5, and declined thereafter. The kinetics of in vivo appearance of effector CD4+ T cells that produce lymphokines upon restimulation in vitro were similar for each of the lymphokines examined. Mice depleted of precursor CD4+ T cells by adult thymectomy exhibited limited capacity to generate lymphokine secreting CD4+ T cells in response to primary immunization with KLH, suggesting that the majority of lymphokine producing T cells arise from short-lived and/or precursor cells. Separation of CD4+ T cells from KLH-primed mice on the basis of expression of the lymph node-specific homing receptor, MEL-14, revealed that antigen-specific production of IL-2, IL-3, IL-4, and IFN-gamma was exclusively associated with the MEL-14- subset of CD4+ T cells. Separation on the basis of CD45RB expression, demonstrated that antigen-specific lymphokine production was primarily associated with the minor CD45RB- population, which has been previously associated with memory activity. Our results indicate that primary in vivo immunization leads to the development of a transient population of helper-effectors with a unique phenotype that can produce large quantities of lymphokines and mediate excellent helper activity for B cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Interferon gamma (IFN-gamma) is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice.

Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-gamma, plays a role in the development of EAMG, we immunized IFN-gamma knockout (IFN-gko) (-/-) mice and wild-type (WT) (+/+) mice of H-2(b) haplotype with AChR in CFA. We observed that AChR-primed lymph node cells from IFN-gko mice proliferated normally to AChR and to its dominant pathogenic alpha146-162 sequence when compared with these cells from the WT mice. However, the IFN-gko mice had no signs of muscle weakness and remained resistant to clinical EAMG at a time when the WT mice exhibited severe muscle weakness and some died. The resistance of IFN-gko mice was associated with greatly reduced levels of circulating anti-AChR antibody levels compared with those in the WT mice. Comparatively, immune sera from IFN-gko mice showed a dramatic reduction in mouse AChR-specific IgG1 and IgG2a antibodies. However, keyhole limpet hemocyanin (KLH)-priming of IFN-gko mice readily elicited both T cell and antibody responses, suggesting that IFN-gamma regulates the humoral immune response distinctly to self (AChR) versus foreign (KLH) antigens. We conclude that IFN-gamma is required for the generation of a pathogenic anti-AChR humoral immune response and for conferring susceptibility of mice to clinical EAMG.

Lymphocyte migration into tissue: the paradigm derived from CD4 subsets.

The appropriate recirculation and migration of naive, effector and memory T cells into inflamed tissue are precisely controlled by adhesive interactions with vascular endothelium. Analyses of CD4 lymphocytes have indicated that naive and antigen-experienced cells exhibit distinctive patterns of homing and recirculation, and that subsets of cells preferentially localize in different anatomical locations as a consequence of previous antigen exposure and differences in adhesion receptor usage.

Correlation of in vitro and in vivo growth suppression of MCF-7 human breast cancer by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The purpose of this study was to compare the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the in vitro and in vivo 17 beta-estradiol (E2)-dependent growth of MCF-7 human breast cancer cells. In culture, a major component of postconfluent growth of MCF-7 cells is E2 dependent. In vivo, MCF-7 cells fail to grow as xenografts without exogenous E2 support. Thus the effect of TCDD on postconfluent MCF-7 growth in culture was compared with its effect on MCF-7 xenograft growth in immunosuppressed mice. A concentration of 10(-9) M E2 was optimal for supporting postconfluent growth of MCF-7 cells in culture into multicellular aggregates (foci) on a monolayer background. The 50% inhibitory dose of TCDD under these conditions was 3 x 10(-10) M, while E2-dependent focus development was completely inhibited by 10(-8) M TCDD. Weekly i.p. administration of TCDD (5 micrograms/kg) to mice bearing MCF-7 tumor xenografts resulted in inhibition of the tumor growth rate for the first 2 weeks, followed by recovery to the control growth rate during the third week. These recovered tumors were found to retain estrogen-dependent growth as shown by second generation transplantation studies. The p.o. route of TCDD administration yielded a similar 2-week transient suppression of growth with a concentration of 8 micrograms TCDD/kg body weight but only a 1-week growth rate latency with a 2-microgram/kg body weight dose. A single 5-micrograms/kg dose given 1 day after implantation was virtually noninhibitory. These results indicate that TCDD suppression of estrogen-dependent MCF-7 human breast cancer cell growth in vitro was predicative of a similar growth suppression of MCF-7 solid tumor xenografts in vivo. However, additional host-related factors must be involved in vivo, since suppression of tumor growth is transient. These studies provide a basis for future in vivo investigations of TCDD endocrine toxicity by using the MCF-7 tumor as a surrogate estrogen-responsive human organ and to examine the efficacy of TCDD and related Ah receptor-mediated compounds in the management of human estrogen-dependent breast cancer.

Gene regulation in an MCF-7 cell line that naturally expresses an estrogen receptor unable to directly bind DNA.

In the described studies, we have developed a variant of the MCF-7 cell line, M-ERd3/g8, for analysis of 17-beta-estradiol (E2)-action without direct DNA interaction by E2-receptors. M-ERd3/g8 cells principally express the estrogen receptor alpha (ER) form ERDelta3 which, due to skipping of exon 3, lacks the second zinc finger of ER that is required for direct DNA interaction. This was achieved by introduction of siRNA targeting exon 3 to a Tamoxifen-selected MCF-7 variant, TMX 2-11, expressing approximately equal amount of full-length ER and ERDelta3 proteins. M-ERd3/g8 cells exhibited a normal nuclear ER localization, and ERDelta3 expression levels were similar to those for full-length ER protein in MCF-7 cells. Ser 118 phosphorylation of the ERDelta3 was triggered by E2 treatment. The expression of several well characterized E2-responsive markers was strongly modified in the M-ERd3/g8 cells. The E2-induction of progesterone receptor (PR) and HEM45 mRNAs was abolished. The effect on pS2 mRNA expression was complex: the pS2 mRNA levels fell approximately 50-fold in control M-ERd3/g8 cells. There was E2-induction of pS2-expression but with an altered temporal pattern. This was blocked by inhibitors of the p42/44 mitogen activated protein (MAP) kinase and inositol triphosphate (PI3) kinase pathways suggesting a role for cytoplasmic signaling pathways. Gene array analysis and real-time polymerase chain reaction (PCR) studies identified several genes whose expressions were induced in E2-treated M-ERd3/g8 cells. These included A-Myb, a homolog to the avian myoblastosis virus oncogene, carbonic anhydrase XII (CAXII), chemokine ligand 12 (CXCL-12), early growth response 3 (EGR 3), fibrinogen B beta (FibBbeta), along with serine protease 23 (SPUVE). The responses fell into several temporal patterns. A-Myb, CAXII, CXCL-12 and EGR 3 were E2-induced within 2 h. The expression of CXCL-12 and EGR 3 was persistent to 24 h, while that of A-Myb and CAXII was not persistent in M-ERd3/g8 cells. FibBbeta and SPUVE expression was not induced until times later than 6 h. Expression of none of the genes was elevated prior to 2 h, but the utilization of a 24 h time point for the gene array analysis may have eliminated the most transiently responsive genes. Immediate early 3 (IE3) was down-regulated by E2 in the M-ERd3/g8 cells but was transiently up-regulated during the 2-6 h period in MCF-7 cells. Basal levels of several of the genes were strongly reduced in M-ERd3/g8, compared to MCF-7. The studies suggest that M-ERd3/g8 cells provide a new model for studies of E2-action without direct ER binding to DNA and where E2-action must be via alternate pathways.

Flow cytometric analysis of I-J expression on murine bone marrow-derived macrophages.

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of development and function of memory CD4 subsets.

Immunologic memory refers to the dramatic response to previously encountered antigen (Ag) that is largely controlled by CD4 T cells. Understanding how CD4 memory is regulated is essential for exploiting the immune system to protect against disease and to dampen immunopathology in allergic responses and autoimmunity. Using defined adoptive-transfer models, we are studying parameters that affect differentiation of memory CD4 cells in vivo and have found that a complex interplay of T cell receptor signaling, costimulation, and cytokines can determine the extent of memory development and the balance of Th1 and Th2 memory subsets. On challenge, memory CD4 cells localize in sites of Ag exposure and develop into effectors that regulate memory responses. We are investigating the roles of adhesion molecules, cytokines, and chemokines in the selective recruitment of CD4 memory subsets to address mechanisms by which memory T cells provide long-lasting immunity and, in our recent studies, to determine how memory CD4 cells contribute to the development of autoimmune diabetes.

Anticoagulation therapy in children with mechanical prosthetic cardiac valves.

From 1980 through 1984, 28 children younger than 19 years (mean 7.9) underwent cardiac valve replacement with 30 mechanical prostheses. Patients were followed for a total of 471 months (mean 15.7) and received either warfarin (mean 0.16 mg/kg/day) or acetylsalicylic acid and dipyridamole (mean 6.1 and 1.9 mg/kg/day, respectively) as thromboembolism prophylaxis. The frequency and incidence of thromboembolism and hemorrhage were compared. Warfarin-treated patients were at increased risk of hemorrhage (5 of 20 [25%], or 22 per 100 patient-years, vs 0 of 10 [0%], or 0 per 100 patient-years, p less than 0.05). Three of the 5 hemorrhagic episodes were mild, and in no case was hemorrhage life-threatening. Patients who did not receive warfarin had a greater risk of thromboembolism (2 of 10 [20%], or 12 per 100 patient-years, vs 0 of 20 [0%], or 0 per 100 patient-years, p less than 0.05). Both episodes of thromboembolism were life-threatening and necessitated emergency valve replacement. Although warfarin is associated with greater risk of hemorrhage than is acetylsalicylic acid and dipyridamole, warfarin is better than antiplatelet drugs in thromboembolism prophylaxis and is indicated for anticoagulation therapy in children with mechanical cardiac prostheses.

Costs and results of cardiac operations in infants less than 4 months old. Are they worthwhile?

From 1979 through 1983, 328 of 1,388 pediatric cardiac operations involved patients undergoing their first procedure at less than 4 months of age. Of these, 220 patients had 265 nonductal procedures, and their case histories are reviewed for results and total hospital cost. Initial operative mortality was 20% (43 patients). Infants with lower operative age and operative weight tended to have closed procedures. Mortality and cure were not related to gestational age, birth weight, age at operation, number of operations, or type of operation. Lower operative weight was associated with a greater mortality. Evaluated survivors (142 patients) were followed for a mean of 24 months. Fifteen percent (33 patients) died during follow-up. Of survivors, 80% (114 patients) had optimized general health; a subset of 29% had normal cardiac function, and 17% were cured. Lower birth weight was associated with curable lesions and normalcy (p less than 0.04). Longer preoperative hospital stay and lower weight at operation were associated with higher hospital cost (p less than 0.05). Hospital cost was not related to type of operation, gestational age, birth weight, age at operation, mortality, cure, or normalcy. Acquired neurologic dysfunction and long-term disability were uncommon. The mean hospital cost for surviving infants was +80,000 (1984 dollars). Effective hospital cost per survivor was +110,000. Mortality, cure, and normal function after cardiac operations in infants less than 4 months of age were not related to gestational age, birth weight, or age at operation. Mortality was higher in patients with a lower weight at operation. Separation into distinct fiscal cost groups is not reasonable in this series. Because most survivors are in normal or optimized cardiac health, intensive cardiovascular care in this population is justified.

Rapid detection of viruses of the tick-borne encephalitis virus complex by RT-PCR of viral RNA.

Studies were performed to identify a pair of primers, specific for the tick-borne encephalitis (TBE) virus complex of the Flaviviridae, with which to develop a rapid and specific identification system based on reverse transcription and the polymerase chain reaction (RT-PCR). The specificity of a putative primer pair was examined by RT-PCR of representative viruses from other antigenic complexes of the Flaviviridae and by computer sequence homology checks. All viruses of the TBE complex tested, with a single exception, were identified by RT-PCR using the identified primer pair. Accumulated data suggest that one of the putative primers identified in these studies may have flavivirus group specificity. The advantages of such a primer in the development of identification systems for all virus complexes of the Flaviviridae is discussed.

Cardiovascular effects of platelet-activating factor.

Sudden release of platelet-activating factor (PAF) into the circulation can cause hypotension, tachycardia, and circulatory collapse. To further examine this response, we performed detailed studies of cardiovascular function after PAF administration to young domestic pigs and newborn piglets. Our results indicate that circulatory dysfunction after PAF reflects severe constriction of pulmonary resistance vessels and consequent acute right ventricular failure. Although PAF-induced coronary artery constriction and contractile depression may be complicating problems, left ventricular underperfusion and dysfunction after PAF are mainly the result of systemic arterial hypotension and diminished left ventricular filling. The adverse hemodynamic effects of PAF are accompanied by substantial release of thromboxane A2 (TxA2). These effects are mimicked by the TxA2 agonist U-46619 and partially blocked by specific and nonspecific inhibitors of TxA2 synthesis (OKY-046 and indomethacin). Even more potent blockade of PAF action is exerted by the TxA2 receptor blocker, SQ 29,548. Taken together, these findings indicate that severe pulmonary vascular constriction and hemodynamic collapse soon after intravenous PAF are at least partially mediated by PAF-induced TxA2 release. Tachyphylaxis to PAF influence has been observed in studies of leukocyte and platelet function. We hypothesized that tachyphylaxis to PAF might also occur in our studies of constrictor responses in pulmonary vessels. Recently, we have examined the capacity of PAF to produce sustained pulmonary vasoconstriction in open-chested, anesthetized newborn piglets. Infusions sufficient to produce 100% increase in mean pulmonary artery pressure after 3 min showed no loss of efficacy when sustained for 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Problems with rigid seed strand lodging during prostate implantation: a proposed mechanism and solution.

Since the introduction of rigid strands of radioactive seeds embedded in absorbable suture material, many brachytherapists have experienced problems with the strands lodging inside the implant needle during the deposition process. By using a scanning electron microscope, we examined some potential factors which could lead to this problem. It seems plausible that the lodging is due to two factors: prostate tissue hindering the motion of the strand initially, and friction between the strand braids and the inner surface of the needle. Both result in an "accordion effect" as the stylet applies pressure on the strand. Based on this assumption, a solution was found. A combination of using needles with a polished inner surface, and repeated clockwise and counterclockwise 360 degrees twisting of the needle about the stylet during the deposition process allows for smooth deposition of the strand at the intended location. By using this technique, one is able to exploit the potential dosimetric advantages of rigid seed strand implants without additional problems.

Pharmacological induction of pancreatic islet cell transdifferentiation: relevance to type I diabetes.

Type I diabetes (T1D) is an autoimmune disease in which an immune response to pancreatic ß-cells results in their loss over time. Although the conventional view is that this loss is due to autoimmune destruction, we present evidence of an additional phenomenon in which autoimmunity promotes islet endocrine cell transdifferentiation. The end result is a large excess of δ-cells, resulting from α- to ß- to δ-cell transdifferentiation. Intermediates in the process of transdifferentiation were present in murine and human T1D. Here, we report that the peptide caerulein was sufficient in the context of severe ß-cell deficiency to induce efficient induction of α- to ß- to δ-cell transdifferentiation in a manner very similar to what occurred in T1D. This was demonstrated by genetic lineage tracing and time course analysis. Islet transdifferentiation proceeded in an islet autonomous manner, indicating the existence of a sensing mechanism that controls the transdifferentiation process within each islet. The finding of evidence for islet cell transdifferentiation in rodent and human T1D and its induction by a single peptide in a model of T1D has important implications for the development of ß-cell regeneration therapies for diabetes.