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Blood smears analysis: a valuable tool for profiling circulating erythrocytes and leukocytes in mouse model of leishmaniasis
Abstract
Although blood smear analysis can yield valuable insights into the health status of laboratory animals, it is not frequently performed in studies involving mouse models of leishmaniasis. Thus, to underscore its importance, we conducted microscopic evaluation of blood smears prepared from 10 µL of blood from L. (L.) amazonensis-infected BALB/c mice. Subsequently, we explored potential correlations between the identified hematological alterations, clinical evaluation data, and serum levels of total proteins (TP), albumin (Alb), globulins (Glob), IL-17A, and IL-10. Our findings revealed that infected mice exhibited an increased erythrocyte count, alongside a higher prevalence of microcytic and hypochromic erythrocytes. Moreover, it was observed elevated relative and absolute numbers of neutrophils and monocytes and reduced relative and absolute numbers of lymphocytes. A significant proportion of monocytes and lymphocytes in infected animals displayed reactive features. The relative number of lymphocytes was negatively correlated with the serum levels of Glob. Furthermore, the frequency of reactive lymphocytes was positively correlated with the serum levels of Glob and IL-17A. There was also a positive correlation between the absolute number of neutrophils and the serum levels of IL-17A and the frequency of reactive monocytes and the serum levels of Glob. Therefore, these findings, collectively, underscore the importance of blood smear analysis for elucidating both the quantitative and qualitative hematological changes experimentally induced by Leishmania infection. Integration of this technique into research protocols involving different mouse models of leishmaniasis holds immense potential for advancing our knowledge of this infectious disease and enhancing clinical management strategies.
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Leishmania parasites are heavily dependent on efficient iron acquisition from a tightly regulated host iron pool for survival and virulence. Prior studies uncovered multiple strategies adopted by the parasite to hijack the iron-regulatory network of macrophages. Despite these extensive studies with infected macrophages, there is limited knowledge of the effect of Leishmania infection on systemic iron homeostasis. This issue is particularly relevant for Leishmania major, which causes localized skin infection with minimal lymphatic spread. We show for the first time that L. major infection in the mouse footpad induced influx of iron at the site of infection through blood with simultaneous upregulation of transferrin receptor 1 (TfR1) and downregulation of phagolysosomal iron exporter Nramp1 expression in the footpad tissue. Interestingly, localized L. major infection had far-reaching effects beyond the infection site triggering anemia-like symptoms. This was evident from depleted physiological iron stores from the liver and bone marrow as well as reduced hemoglobin levels and deformed erythrocytes. The infected mice also developed splenomegaly with signs of splenic stress erythropoiesis as indicated by upregulation of several erythroid-related genes. These observations prompted us to provide oral iron supplementations to the L. major infected mice, which resulted in a drastic reduction of the parasite load and restoration of iron homeostasis.
Visceral leishmaniasis is an important yet neglected parasitic disease caused by infection with Leishmania donovani or L infantum. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulation, and extensive hematological complications. Thrombocytopenia is a dominant hematological feature seen in both humans and experimental models, but the mechanisms behind this infection-driven thrombocytopenia remain poorly understood. Using a murine model of experimental visceral leishmaniasis (EVL), we demonstrated a progressive decrease in platelets from day 14 after infection, culminating in severe thrombocytopenia by day 28. Plasma thrombopoietin (TPO) levels were reduced in infected mice, at least in part because of the alterations in the liver microenvironment associated with granulomatous inflammation. Bone marrow (BM) megakaryocyte cytoplasmic maturation was significantly reduced. In addition to a production deficit, we identified significant increases in platelet clearance. L donovani–infected splenectomized mice were protected from thrombocytopenia compared with sham operated infected mice and had a greater response to exogenous TPO. Furthermore, infection led to higher levels of platelet opsonization and desialylation, both associated with platelet clearance in spleen and liver, respectively. Critically, these changes could be reversed rapidly by drug treatment to reduce parasite load or by administration of TPO agonists. In summary, our findings demonstrate that the mechanisms underpinning thrombocytopenia in EVL are multifactorial and reversible, with no obvious residual damage to the BM microenvironment.
An evaluation of mouse red blood cell (RBC) and platelet (PLT) counting with an automated hematology analyzer was performed with three strains of mice, C57BL/6 (B6), BALB/c (BALB) and DBA/2 (D2). There were no significant differences in RBC and PLT counts between manual and automated optical methods in any of the samples, except for D2 mice. For D2, RBC counts obtained using the manual method were significantly lower than those obtained using the automated optical method (P<0.05), and PLT counts obtained using the manual method were higher than those obtained using the automated optical method (P<0.05). An automated hematology analyzer can be used for RBC and PLT counting; however, an appropriate method should be selected when D2 mice samples are used.
Hemophagocytosis is a phenomenon in which macrophages phagocytose blood cells. There are reports on up-regulated hemophagocytosis in patients with infectious diseases including typhoid fever, tuberculosis, influenza and visceral leishmaniasis (VL). However, mechanisms of infection-associated hemophagocytosis remained elusive due to a lack of appropriate animal models. Here, we have established a mouse model of VL with hemophagocytosis. At 24 weeks after infection with 1 x 107 Leishmania donovani promastigotes, BALB/cA mice exhibited splenomegaly with an average tissue weight per body weight of 2.96%. In the tissues, 28.6% of macrophages contained phagocytosed erythrocytes. All of the hemophagocytosing macrophages were parasitized by L. donovani, and higher levels of hemophagocytosis was observed in heavily infected cells. Furthermore, more than half of these hemophagocytes had two or more macrophage-derived nuclei, whereas only 15.0% of splenic macrophages were bi- or multi-nuclear. These results suggest that direct infection by L. donovani causes hyper-activation of host macrophages to engulf blood cells. To our knowledge, this is the first report on hemophagocytosis in experimental Leishmania infections and may be useful for further understanding of the pathogenesis.
Visceral Leishmaniasis (VL) is an endemic parasitic disease and remains as a major health concern in southwestern Iran. The current study describes clinico-hematological, epidemiological and therapeutic features of VL cases, admitted to university-affiliated hospitals, during 1999-2014 in Fars province, southwestern Iran. A total of 380 VL cases were recorded during a 16 years period, giving an average annual admission of 23.75 cases/year in which 217 (57.1%) were male and 163 (42.9%) were female. Mean age of the patients was 3.7 years. The majority of the cases (91.5%) were ≤ 5 years old. Bone-marrow aspiration detected Leishmania amastigotes only in 26.6% of cases. Fever (98.1%), abdominal protrusion (65.1%) and hepatosplenomegaly (63.7%) were the most common clinical presentations of the patients. Pancytopenia was noted in 43.1, anemia in 87.3 and thrombocytopenia in 64% of cases. Increase in the level of AST (aspartate aminotransferase), ALT (alanine aminotransferase), alkaline phosphatase, LDH (lactate dehydrogenase) and CRP (C-Reactive Proteins) were seen in 84.9, 53.6, 44.4, 72.5 and 83.1% of cases, respectively. Mortality was noted in 5.3% of cases. Deranged haemato-biochemical parameters including total and direct bilirubin, PLT (platelet) and pancytopenia were significantly contributed to mortality from VL. Moreover, clinical features such as severe splenomegaly as well as bacterial infections were meaningfully contributed to death from VL. The majority of patients (74.9%) were treated with meglumine antimoniate. Amphotericin B was administrated in 59 of cases, 11 of them were initially treated with meglumine antimoniate with a shift to amphotericin B, because of treatment failure. Findings of the current study demonstrated that VL is present in southwest of Iran with a fairly continual rate during the last 16 years period. Deranged haemato-biochemical parameters along with severe splenomegaly contributed to mortality from VL.
Adult stem cells have a great potential applicability in regenerative medicine and cell-based therapies. However, there are still many unresolved issues concerning their biology, and the influence of the local microenvironment on properties of stem cells has been increasingly recognized. Interleukin (IL-) 17, as a cytokine implicated in many physiological and pathological processes, should be taken into consideration as a part of a regulatory network governing tissue-associated stem cells’ fate. This review is focusing on the published data on the effects of IL-17 on the properties and function of hematopoietic and mesenchymal stem cells and trying to discuss that IL-17 achieves many of its roles by acting on adult stem cells.
Hematologic parameters are important markers of disease in human and veterinary medicine. Biomedical research has benefited from mouse models that recapitulate such disease, thus expanding knowledge of pathogenetic mechanisms and investigative therapies that translate across species. Mice in health have many notable hematologic differences from humans and other veterinary species, including smaller erythrocytes, higher percentage of circulating reticulocytes or polychromasia, lower peripheral blood neutrophil and higher peripheral blood and bone marrow lymphocyte percentages, variable leukocyte morphologies, physiologic splenic hematopoiesis and iron storage, and more numerous and shorter-lived erythrocytes and platelets. For accurate and complete hematologic analyses of disease and response to investigative therapeutic interventions, these differences and the unique features of murine hematopathology must be understood. Here we review murine hematology and hematopathology for practical application to translational investigation.
Visceral leishmaniasis (VL) is one of the most important parasitic diseases endemic in northwestern and southern areas of Iran. The aim of the present study was to review the records of children hospitalized with VL in order to characterize the clinical features of children as well as laboratory finding in Children Medical Center Hospital, Tehran, Iran.
The medical records of all children with a final diagnosis of VL were reviewed from 2004 to 2011. Demographic, clinical information, laboratory finding and treatment were considered.
A total number of 34 children with confirmed VL through 2004-2011 were included in the study. The most prevalent sign and symptoms were fever (97.1%), pallor and weakness (97.1%), appetite loss (61.8%), splenomegaly (97.1%) and hepatomegaly (88.2%). The most frequent laboratory abnormalities were hematological including anemia (97.1%), thrombocytopenia (91.2%) and leukopenia (67.6%). Direct agglutination test (DAT) was performed in 23 cases and all of them showed anti-Leishmania antibodies with titers of ≥ 1: 3200. In addition, 90% of patients had positive rK39 results. Identification of Leishmania in the aspirates of the bone marrow was found in 83.3% of patients.
Regional surveillance system in order to monitoring of leishmaniasis trends as well as detection of new emerging foci is recommended.
Rheumatoid arthritis (RA) is a chronic inflammatory disease that is mediated, in part, by proinflammatory factors produced by RA synovial tissue (ST) fibroblasts and macrophages, resulting in monocyte migration from the blood to the ST. To characterize the potential role of IL-17 in monocyte migration, RA synovial fibroblasts and macrophages were activated with IL-17 and examined for the expression of monocyte chemokines. The two potentially important monocyte chemoattractants identified were CCL20/MIP-3alpha and CCL2/MCP-1, which were significantly induced in RA synovial fibroblasts and macrophages. However, in vivo, only CCL2/MCP-1 was detectable following adenovirus IL-17 injection. We found that IL-17 induction of CCL2/MCP-1 was mediated by the PI3K, ERK, and JNK pathways in RA ST fibroblasts and by the PI3K and ERK pathways in macrophages. Further, we show that neutralization of CCL2/MCP-1 significantly reduced IL-17-mediated monocyte recruitment into the peritoneal cavity. We demonstrate that local expression of IL-17 in ankle joints was associated with significantly increased monocyte migration and CCL2/MCP-1 levels. Interestingly, we show that RA synovial fluids immunoneutralized for IL-17 and CCL2/MCP-1 have similar monocyte chemotaxis activity as those immunoneutralized for each factor alone. In short, CCL2/MCP-1 produced from cell types present in the RA joint, as well as in experimental arthritis, may be responsible, in part, for IL-17-induced monocyte migration; hence, these results suggest that CCL2/MCP-1 is a downstream target of IL-17 that may be important in RA.
CD4+ T cells, upon activation and expansion, develop into different T helper cell subsets with different cytokine profiles and distinct effector functions. Until recently, T cells were divided into Th1 or Th2 cells, depending on the cytokines they produce. A third subset of IL-17-producing effector T helper cells, called Th17 cells, has now been discovered and characterized. Here, we summarize the current information on the differentiation and effector functions of the Th17 lineage. Th17 cells produce IL-17, IL-17F, and IL-22, thereby inducing a massive tissue reaction owing to the broad distribution of the IL-17 and IL-22 receptors. Th17 cells also secrete IL-21 to communicate with the cells of the immune system. The differentiation factors (TGF-beta plus IL-6 or IL-21), the growth and stabilization factor (IL-23), and the transcription factors (STAT3, RORgammat, and RORalpha) involved in the development of Th17 cells have just been identified. The participation of TGF-beta in the differentiation of Th17 cells places the Th17 lineage in close relationship with CD4+CD25+Foxp3+ regulatory T cells (Tregs), as TGF-beta also induces differentiation of naive T cells into Foxp3+ Tregs in the peripheral immune compartment. The investigation of the differentiation, effector function, and regulation of Th17 cells has opened up a new framework for understanding T cell differentiation. Furthermore, we now appreciate the importance of Th17 cells in clearing pathogens during host defense reactions and in inducing tissue inflammation in autoimmune disease.
The efficacy of two mesoionic derivatives (MI-H-H and MI-4-OCH(3)) was evaluated in CBA/J mice infected with Leishmania amazonensis. Treatment with these compounds demonstrated that the MI-4-OCH(3) derivative and the reference drug meglumine antimoniate (Glucantime) presented significant activity relative to an untreated control. No apparent hepatic or renal toxicity due to these mesoionic compounds was found.
The effect of blood sampling site on the hemogram and neutrophil adhesion molecules was examined in BALB/c mice.
Blood samples were drawn from the tail, eye, and heart during anesthesia with ketamine and xylazine. Cell numbers were quantified with an automated counter and flow cytometry was used to quantify CD11b and CD18.
Total white blood cell (WBC) counts were highest from tail, lower from eye, and significantly lower from heart blood. In general, differences between tail and heart counts reflected changes in all cell types. RBCs, platelets and hematocrits were significantly increased in tail compared to heart blood. Although CD18 levels were not different, CD11b was significantly higher on neutrophils from tail compared to heart blood.
In anesthetized BALB/c mice, sampling site readily influences blood counts and neutrophil CD11b. The findings underscore the need to standardize sampling site when measuring these parameters.
Abstract
Background: We aimed to determine the cellular recruitment (leukocyte rolling
and adhesion) by which the Leishmania (Viannia) braziliensis, L. (Leishmania) amazonensis, and L. (Leishmania) major species in the mesenteric microcirculation of
BALB/c mice.
Methods: Five experimental groups were considered: group 1 (L. braziliensis);
group 2 (L. amazonensis); group 3 (L. major); group 4 (control group with PBS);
group 5 (negative control group), analyzed 3, 6, 12, and 24 h after parasite inoculation.
Results: Infections by the different Leishmania species caused an increase in the
number of rolling leukocytes: L. braziliensis a peak at 6 h; L. amazonensis and L. major a peak at 3 h. The Leishmania infections induced leukocyte adhesion: L. major
and L. amazonensis showed an increase after 3 and 6 h, respectively.
Conclusion: The kinetics of cellular recruitment in Leishmania infections, leading
to infection susceptibility or resistance, indicates that distinct mechanisms regulate
the initial response to Leishmania infection and determine its course.
Two approaches can be used to help generate data more quickly from pathology specimens and in a form that can be more easily statistically manipulated: (i) use standardized terminology for the description of lesions, and (ii) apply scoring or quantitative methodology to enrich the data. The usefulness of these techniques is greatly assisted by consistent sample selection and tissue trimming patterns. It is important that the expectations for the histopathology results are discussed with the pathologist before tissue blocks and sections are made so that the selection and trimming of tissues can be optimized before embedding. Reduction in numbers or absence of controls may seem attractive from a 3Rs (reduce, replace, refine) perspective or to reduce the costs of histological and pathological analysis but may be unjustifiable if an experiment ultimately needs to be repeated because of uninterpretable or equivocal data.
INTRODUCTION: Visceral Leishmaniasis is the most severe form of leishmaniasis and can be fatal in the absence of treatment. Nepal, India, Bangladesh, Brazil and Sudan constitute five countries of the world where more than 90% of visceral leishmaniasis occurs. The aim of this study is to evaluate haematological profile with available clinical data in visceral leishmaniasis patients and to detect LD bodies among them. MATERIALS AND METHODS: It is a hospital based cross sectional study conducted in the Department of Pathology, BPKIHS, Dharan, for the period of one year. LD bodies were calculated in bone marrow aspirate of forty clinically suspected cases by counting the number of parasites per 100 consecutive oil immersion fields. RESULTS: The age ranged from 2-60 years. Pyerxia was the most common sign (100%) followed by splenomegaly (82.5%), hepatomegaly (65%), and pallor (75%). Anemia was present in 90%, leucopenia in 67.5% and thrombocytopenia in 72.5% cases. Bicytopenia and pancytopenia were observed in 40% and 25% cases, respectively. On peripheral examination RBCs were predominantly normocytic normochromic. On bone marrow examination normocellular marrow and megaloblastic features were predominant findings followed by increased plasma cells. Low, moderate and high grade LD bodies were present in 7.5%, 37.5% and 55% of the cases respectively. Hepatomegaly, anemia, neutropenia and lymphocytosis were statistically significant to parasite load (p-value <0.05). CONCLUSIONS: Besides LD bodies in bone marrow aspirates, dyserythroblastic changes and increase plasma cells are common findings in leishmaniasis. Patient from endemic area with positive clinical history and findings should be examined for LD bodies in marrow if dyserythroblastic and increase plasma cell picture is found. DOI: http://dx.doi.org/10.3126/ijim.v2i2.8320 Int J Infect Microbiol 2013;2(2):39-44
Laboratory mice, offspring of CB6 F1 s, were maintained at a constant temperature of 21.0 ± 1.0° C and in constant light (LL) of 40 lux or under 12 h of light (40 lux) alternating with 12 h of darkness (LD) for 8 consecutive days. During the following 36 h, tail blood was secured from each mouse at 6-h intervals. From those samples, a differential white blood cell count, based on 100 leukocytes, was made for each animal. The relative numbers of eosinophils, basophils, and monocytes did not fluctuate rhythmically under LL or LD. The per cent distributions of lymphocytes and neutrophils did vary with circadian frequency in both LL and LD (Figs. 1, 2) The peak counts of neutrophils were found in blood sampled near midnight; high counts of lymphocytes were made in smears prepared about 6:00 a.m. and 6:00 p.m.
Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite may cause visceral leishmaniasis, which can also be transmitted to humans. Infected symptomatic dogs show disorganization in the white pulp in spleen tissue and a reduction in T lymphocytes in peripheral blood. To investigate whether apoptosis is involved in white pulp disorganization and diminished T cell counts in peripheral blood, apoptotic T cells from the spleen and peripheral blood of dogs naturally infected with L. (L.) chagasi and presenting clinical manifestations were quantified and compared with healthy dogs. Thirteen symptomatic adult dogs infected by L. (L.) chagasi and six healthy dogs from a nonendemic area (controls) were included in the study. Samples from spleen and peripheral blood were used to quantify apoptosis in CD3 lymphocytes by flow cytometry using Anexin V and Multicaspase kits; the results were compared using the Mann Whitney test. The percentage of total T cells was lower in Leishmania infected dogs compared to healthy controls (P<0.05). Apoptosis levels in T cells from PBMC and spleen were higher in infected dogs than in controls (P<0.05). The least squares method test was used to determine the effect between the degree of structural organization of spleen white pulp and the percentage of apoptosis in the spleen. A significant effect on the level of white pulp morphological disorganization and percentage of apoptosis in spleen T cells was observed (F=20.45; P=0.0014). These data suggest that apoptosis is an important for the immunopathogenesis of canine visceral leishmaniasis.
Visceral Leishmaniasis (VL) or Kala Azar is a chronic infectious disease caused by parasites of the Leishmania donovani complex that can cause various hematologic manifestations. It is characterized by fever, enlargement of liver and spleen, weight loss, pancytopenia and hypergammaglobinemia. It is endemic in the Indian subcontinent, mainly seen in the states of Bihar and West Bengal. Patients with VL can present to the haematologist for various haematological problems prior to receiving the diagnosis of VL. Anaemia is the most common haematological manifestation of VL. VL may also be associated with leucopenia, thrombocytopenia, pancytopenia, hemophagocytosis and disseminated intravascular coagulation. Hematological improvement is noted within a week and complete hematological response occurs in 4-6 weeks of treatment. Relapses are rare and increased risk of being diagnosed with hematolymphoid malignancies on long term follow up is not noted.
The visceral and lethal infection produced in BALB/c mice by Leishmania tropica (major) is accompanied by splenomegaly, anaemia and reversal of albumin-to-globulin ratio. The percentages of both B and T cells are decreased in the spleen. The spleen and lymph nodes become populated with large Ig-, Thy 1.2- 'null' cells. The similarity of some of these parameters with those produced in human kala-azar is discussed.
Anaemia of chronic disease (ACD), the most frequent anaemia among hospitalized patients, develops under chronic inflammatory disorders such as chronic infections, cancer or autoimmune diseases. A number of different pathways contribute to ACD, such as diversion of iron traffic, a diminished erythropoiesis, a blunted response to erythropoietin, erythrophagocytosis and bone marrow invasion by tumour cells and pathogens. Nevertheless, ACD is a reflection of an activated immune system and possibly results from an innovative defence strategy of the body in order to withdraw the essential growth factor iron from invading pathogens and to increase the efficacy of cell-mediated immunity. Diagnosis of ACD can be assessed by examination of chances in serum iron parameters with low to normal serum iron, transferrin saturation and transferrin concentrations on the one hand and normal to increased ferritin, zinc protoporphyrin IX and cytokine levels on the other side. Therapy of ACD includes the cure of the underlying the disease. Apart from this transfusions for rapid correction of haemoglobin levels, and human recombinant erythropoietin for prolonged therapy are used. However, response rates to recombinant erythropoietin are sometimes low. Iron alone should be strictly avoided due to its growth-promoting effect towards micro-organisms and tumour cells and because of it capacity to inhibit T-cell-mediated immune effector pathways. We urgently need prospective clinical trials to gain knowledge about the effects of anaemia correction and/or the use of erythropoietin towards the course of the underlying disease, to find out if a combination therapy with erythropoietin and iron may be beneficial in ACD and to define therapeutic end-points.
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