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PubMed1991

Recognition of MHC TL gene products by gamma delta T cells.

We have studied the ligand specificity of a gamma delta T-cell receptor (TCR) derived from a mouse T-cell hybridoma (KN6). KN6 cells reacted with syngeneic (C57BL/6) cells from various origins (splenocytes, thymocytes, peritoneal exudate cells, etc.) and cells from many different mouse strains. KN6 reactivity against cells from a panel of congenic and recombinant mouse strains demonstrated that the ligand recognized by KN6 is controlled by an MHC-linked gene that most probably maps in the TL region. We cloned this gene and formally proved that it does map in the TL region. This gene turned out to be a novel class I gene (designated T22b) belonging to a hitherto unidentified cluster of TL region genes in strain C57BL/6. This gene was expressed in many different tissues and cell types. We also examined the tissue expression of several other TL genes. One of these, the structural gene (T3b) encoding the thymus leukemia (TL) antigen from C57BL/6 mice, was specifically expressed in the epithelium of the small intestine. Since the intestinal epithelium of the mouse is known to be the homing site for a subset of gamma delta T cells (i-IEL) bearing diverse TCR with V7 rearranged gamma chains, we propose that the T3b gene product is part of the ligand recognized by some of the i-IEL. Our data support the idea that gamma delta T cells might be specific for non-classical class I or class I-like molecules and suggest that gamma delta TCR and non-classical MHC co-evolved for the recognition of a conserved set of endogenous or foreign peptides.

Age Factors↗
PubMed2000

Antitumor vaccination: where we stand.

BACKGROUND AND OBJECTIVES: Vaccination is an effective medical procedure of preventive medicine based on the induction of a long-lasting immunologic memory characterized by mechanisms endowed with high destructive potential and specificity. In the last few years, identification of tumor-associated antigens (TAA) has prompted the development of different strategies for antitumor vaccination, aimed at inducing specific recognition of TAA in order to elicit a persistent immune memory that may eliminate residual tumor cells and protect recipients from relapses. In this review characterization of TAA, different potential means of vaccination in experimental models and preliminary data from clinical trials in humans have been examined by the Working Group on Hematopoietic Cells. EVIDENCE AND INFORMATION SOURCES: The method employed for preparing this review was that of informal consensus development. Members of the Working Group met four times and discussed the single points, previously assigned by the chairman, in order to achieve an agreement on different opinions and approve the final manuscript. Some of the authors of the present review have been working in the field of antitumor immunotherapy and have contributed original papers to peer-reviewed journals. In addition, the material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline. STATE OF THE ART: The cellular basis of antitumor immune memory consists in the generation and extended persistence of expanded populations of T- and B-lymphocytes that specifically recognize and react against TAA. The efficacy of the memory can be modulated by compounds, called "adjuvants", such as certain bacterial products and mineral oils, cytokines, chemokines, by monoclonal antibodies triggering co-stimulatory receptors. Strategies that have been shown in preclinical models to be efficient in protecting from tumor engraftment, or in preventing a tumor rechallenge, include vaccination by means of soluble proteins or peptides, recombinant viruses or bacteria as TAA genes vectors, DNA injection, tumor cells genetically modified to express co-stimulatory molecules and/or cytokines. The use of professional antigen-presenting cells, namely dendritic cells, either pulsed with TAA or transduced with tumor-specific genes, provides a useful alternative for inducing antitumor cytotoxic activity. Some of these approaches have been tested in phase I/II clinical trials in hematologic malignancies, such as lymphoproliferative diseases or chronic myeloid leukemia, and in solid tumors, such as melanoma, colon cancer, prostate cancer and renal cell carcinoma. Different types of vaccines, use of adjuvants, timing of vaccination as well as selection of patients eligible for this procedure are discussed in this review. PERSPECTIVES: Experimental models demonstrate the possibility of curing cancer through the active induction of a specific immune response to TAA. However, while pre-clinical research has identified several possible targets and strategies for tumor vaccination the clinical scenario is far more complex for a number of possible reasons. Since experimental data suggest that vaccination is more likely to be effective on small tumor burden, such as a minimal residual disease after conventional treatments, or tumors at an early stage of disease, better selection of patients will allow more reliable clinical results to be obtained. Moreover, a poor correlation is frequently observed between the ability of TAA to induce a T-cell response in vitro and clinical responses. Controversial findings may also be due to the techniques used for monitoring the immune status. Therefore, the development of reliable assays for efficient monitoring of the state of immunization of cancer patients against TAA is an important goal that will markedly improve the progress of antitumor vaccines. (ABSTRACT TRUNCATED)

Adjuvants, Immunologic↗
PubMed2000

Perceptual learning for a pattern discrimination task.

Our goal was to differentiate low and mid level perceptual learning. We used a complex grating discrimination task that required observers to combine information across wide ranges of spatial frequency and orientation. Stimuli were 'wicker'-like textures containing two orthogonal signal components of 3 and 9 c/deg. Observers discriminated a 15% spatial frequency shift in these components. Stimuli also contained four noise components, separated from the signal components by at least 45 degrees of orientation or approximately 2 octaves in spatial frequency. In Experiment 1 naive observers were trained for eight sessions with a four-alternative same-different forced choice judgment with feedback. Observers showed significant learning, thresholds dropped to approximately 1/3 of their original value. In Experiment 2 we found that observers showed far less learning when the noise components were not present. Experiment 3 found, unlike many other studies, almost complete transfer of learning across orientation. The results of Experiments 2 and 3 suggest that, unlike many other perceptual learning studies, most learning in Experiment 1 occurs at mid to high levels of processing rather than within low level analyzers tuned for spatial frequency and orientation. Experiment 4 found that performance was more severely impaired by spatial frequency shifts in noise components of the same spatial frequency or orientation as the signal components (though there was significant variability between observers). This suggests that after training observers based their responses on mechanisms tuned for selective regions of Fourier space. Experiment 5 examined transfer of learning from a same-sign task (the two signal components both increased/decreased in spatial frequency) to an opposite-sign task (signal components shifted in opposite directions in frequency space). Transfer of learning from same-sign to opposite-sign tasks and vice versa was complete suggesting that observers combined information from the two signal components independently.

Adult↗
PubMed1989

The role of T cell receptors in non-MHC-restricted cytotoxicity.

The relationship between natural killer cells (NK) and cells of the T lineage has been obscured by the existence of poorly characterized clones of presumed NK origin. We have analyzed nine of these cloned cell lines displaying varying levels of cytotoxic activity against NKS YAC-1 target cells for rearrangement and expression of the genes encoding the alpha, beta, and gamma chains of the T cell receptor for antigen. Rearrangements at both the TcR beta and gamma loci were detected in all clones often at both alleles. Rearrangement patterns at the TcR beta locus were identical in several clones, despite different degrees of cytotoxicity. T cell receptor alpha, beta, and gamma genes were expressed as full length transcripts in all clones regardless of their levels of cytotoxic activity. To explore the involvement of cell surface molecules in the cytolytic events, studies were undertaken to determine whether cytotoxic activity could be inhibited by antibodies against CD3, LFA-I, and H-2KdDd. In two selected clones, both alpha and beta chains of the LFA-I molecule were expressed but only monoclonal antibodies against the alpha chain significantly blocked cytotoxicity. Cytotoxicity was also inhibited by monoclonal antibodies against epitopes of H-2KdDd and CD3, the extent of inhibition correlating with the level of surface expression on both clones. These data suggest that conventional alpha/beta heterodimers may be necessary but not sufficient for target cell recognition by these clones. Since T cell receptor rearrangement and expression occur normally in the T cell lineage but not the NK lineage, these results also indicate that a subpopulation of cells with non-MHC-restricted killer activity lies on the T cell differentiation pathway and is selected by in vitro growth with IL-2. The limited rearrangement pattern observed can be explained if only a small subpopulation of T cells is capable of non-MHC-restricted killing, and if certain rearrangements favor self-MHC recognition which is known to block cytolysis in the NK system.

Animals↗
PubMed2026

Endosymbiotic theory of aging revisited: Age-related leakage of mitochondrial dsDNA/RNA stimulates cytosolic nucleic acid sensors which remodel the immune network and promote the aging process.

About 1.5-2 billion years ago, an endosymbiosis between aerobic α-proteobacteria and anaerobic archaeal cells generated mitochondria, i.e., organelles capable of producing oxidative energy. The bacterial genome was fundamentally reduced and a circular mitochondrial genome evolved containing mainly the genes coding for the subunits of the electron transport chain. Before the symbiotic event, there existed a virus-host co-evolution which involved the development of sensors for detecting dangerous viral DNA/RNA molecules. Endosymbiosis supplied eukaryotic cells not only with an oxidative powerhouse to allow the evolution of more complex multicellular organisms but it also meant that cells now housed an organelle which was able to generate reactive oxygen species (ROS) and to leak mitochondrial DNA (mtDNA) and double-stranded RNA (dsRNA) into the cytoplasm. There is now abundant evidence that during aging and age-related diseases mitochondria are prone to release both mtDNA and dsRNA. In the cytoplasm, mtDNA/dsRNA molecules activate a number of cytosolic nucleic acid sensors leading to the secretion of type-1 interferons (IFN) and many other cytokines which promote an age-related proinflammatory state. Currently, it is known that mtDNA can activate the cGAS-STING pathway, AIM2 inflammasomes, IFI16 receptors, and ZBP1 sensors and in addition mitochondrial dsRNA stimulates RIG-1/MDA5 signaling. Interestingly, there is abundant evidence that all these receptors are drivers of cellular senescence and inflammaging. For decades, there has been mounting evidence that mitochondria have a crucial role in the aging process. We will examine this question from the perspective of evolution and propose that mitochondrial evolution created an endogenic source for the leakage of dangerous mtDNA/dsRNA which subsequently stimulated cytosolic DNA/RNA sensors, an evolutionarily conserved viral defence mechanism. It seems that these two evolutionary events provided not only the basis for the inevitable process of aging but also ensuring the death of parental organisms.

Aging↗
PubMed2003

Lumbar spine visualisation based on kinematic analysis from videofluoroscopic imaging.

Low back pain is a significant problem and its cost is enormous to society. However, diagnosis of the underlying causes remains problematic despite extensive study. Reasons for this arise from the deep-rooted situation of the spine and also from its structural complexity. Clinicians have to mentally convert 2-D image information into a 3-D form to gain a better understanding of structural integrity. Therefore, visualisation and animation may be helpful for understanding, diagnosis and for guiding therapy. Some low back pain originates from mechanical disorders, and study of the spine kinematics may provide an insight into the source of the problem. Digital videofluoroscopy was used in this study to provide 2-D image sequences of the spine in motion, but the images often suffer due to noise, exacerbated by the very low radiation dosage. Thus determining vertebrae position within the image sequence presents a considerable challenge. This paper describes a combination of spine kinematic measurements with a solid model of the human lumbar spine for visualisation of spine motion. Since determination of the spine kinematics provides the foundation and vertebral extraction is at the core, this is discussed in detail. Edge detection is a key feature of segmentation and it is shown that phase congruency performs better than most established methods with the rather low-grade image sequences from fluoroscopy. The Hough transform is then applied to determine the positions of vertebrae in each frame of a motion sequence. In the Hough transform, Fourier descriptors are used to represent the vertebral shapes. The results show that the Hough transform is a very promising technique for vertebral extraction from videofluoroscopic images. A dynamic visualisation package has been developed in order to view the moving lumbar spine from any angle and viewpoint. Wire frame models of the vertebrae were built by using CT images from the Visible Human Project and these models are scaled to match the fluoroscopic image data. For animation, the spinal kinematic data from the motion study is incorporated.

Algorithms↗
PubMed1991

Interactions of bone marrow cells from young and old mice with syngeneic and allogeneic thymic tissue.

Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy1.2 and ARK/J-Thy1.1 seeded onto C57BL/6J FT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy1.2 mixed with C57BL/Ka-Thy1.1 seeded onto C57BL/6J or C57BL/Ka FT), the Thy1+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thmocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice.

Age Factors↗
PubMed1991

Molecular and cellular analysis of human T lymphocytes expressing gamma delta T-cell receptor.

A minor subset of T lymphocytes expresses a CD3-associated TCR composed of gamma and delta chains. The majority of TCR gamma/delta+ cells lack surface CD4 and CD8 antigen and do not react with WT31 mAb. These negative criteria were utilized in early studies to identify TCR gamma/delta+ cells. More recently, mAb to TCR gamma/delta, selected in different laboratories, have permitted the direct identification of TCR gamma/delta+ cells and their subsets. TCR gamma/delta molecules were found to be heterogeneous in size and charge mobility. Two major forms of TCR gamma/delta could be identified that are characterized by the presence or absence of an inter-chain disulphide bond. Biochemical analysis originally suggested that a precise correlation existed between reactivity with BB3 or delta TCS1/A13 mAb and expression of a disulphide (C gamma 1-encoded) or non-disulphide linked (C gamma 2-encoded) form of TCR gamma/delta. However, more recent studies have indicated that these mAb react with the molecular product of V delta 2 or V delta 1, respectively, mAb directed to one or another form of TCR gamma/delta activate the functional program of the cell, leading to intracellular Ca++ mobilization, lymphokine production and triggering of the lytic machinery. Analysis of the target molecules for TCR gamma/delta-mediated recognition revealed that at least some TCR gamma/delta+ cells are capable of specific responses to (allo)antigen and that polymorphic determinants of class I molecules can be recognized (as shown by the specific lysis of P815 cells transfected with HLA-24 allele). Unlike TCR alpha/beta+ cells, TCR gamma/delta+ cells are homogeneously composed of cytolytic precursors, as shown by the analysis of a large panel of clones in both lectin-dependent and redirected killing assays. In spite of their LGL morphology, freshly isolated TCR gamma/delta+ cells do not lyse NK-sensitive targets but do so after exposure to rIL-2. A modest cytolytic activity, however, could be induced also in fresh cells by anti-TCR/CD3 mAb in a redirected killing assay. Analysis of the distribution of the subsets expressing different TCR gamma/delta types showed that BB3+ cells are prevalent in the peripheral blood and virtually absent in the thymus; in contrast, A13+ (delta TCS1+) cells represent the majority of TCR gamma/delta+ thymocytes. Electron microscopic analysis of fresh TCR gamma/delta+ cells showed an extended cytoplasm containing numerous electron-dense granules identifiable as primary lysosomes. Upon stimulation with IL-2, TCR gamma/delta+ cells, similar to other LAK cells, display an increase in their cytoplasmic granules together with a redistribution of cytoskeletal structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed2000

Kawasaki disease: A brief history.

Tomisaku Kawasaki published the first English-language report of 50 patients with Kawasaki disease (KD) in 1974. Since that time, KD has become the leading cause of acquired heart disease among children in North America and Japan. Although an infectious agent is suspected, the cause remains unknown. However, significant progress has been made toward understanding the natural history of the disease and therapeutic interventions have been developed that halt the immune-mediated destruction of the arterial wall. We present a brief history of KD, review progress in research on the disease, and suggest avenues for future study. Kawasaki saw his first case of KD in January 1961 and published his first report in Japanese in 1967. Whether cases existed in Japan before that time is currently under study. The most significant controversy in the 1960s in Japan was whether the rash and fever sign/symptom complex described by Kawasaki was connected to subsequent cardiac complications in a number of cases. Pathologist Noboru Tanaka and pediatrician Takajiro Yamamoto disputed the early assertion of Kawasaki that KD was a self-limited illness with no sequelae. This controversy was resolved in 1970 when the first Japanese nationwide survey of KD documented 10 autopsy cases of sudden cardiac death after KD. By the time of the first English-language publication by Kawasaki in 1974, the link between KD and coronary artery vasculitis was well-established. KD was independently recognized as a new and distinct condition in the early 1970s by pediatricians Marian Melish and Raquel Hicks at the University of Hawaii. In 1973, at the same Hawaiian hospital, pathologist Eunice Larson, in consultation with Benjamin Landing at Los Angeles Children's Hospital, retrospectively diagnosed a 1971 autopsy case as KD. The similarity between KD and infantile periarteritis nodosa (IPN) was apparent to these pathologists, as it had been to Tanaka earlier. What remains unknown is the reason for the simultaneous recognition of this disease around the world in the 1960s and 1970s. There are several possible explanations. KD may have been a new disease that emerged in Japan and emanated to the Western World through Hawaii, where the disease is prevalent among Asian children. Alternatively, KD and IPN may be part of the spectrum of the same disease and clinically mild KD masqueraded as other diseases, such as scarlet fever in the preantibiotic era. Case reports of IPN from Western Europe extend back to at least the 19th century, but, thus far, cases of IPN have not been discovered in Japan before World War II. Perhaps the factors responsible for KD were introduced into Japan after the World War II and then reemerged in a more virulent form that subsequently spread through the industrialized Western world. It is also possible that improvements in health care and, in particular, the use of antibiotics to treat infections caused by organisms including toxin-producing bacteria reduced the burden of rash/fever illness and allowed KD to be recognized as a distinct clinical entity. Itsuzo Shigematsu, Hiroshi Yanagawa, and colleagues have conducted 14 nationwide surveys in Japan. These have indicated that: 1) KD occurred initially in nationwide epidemics but now occurs in regional outbreaks; 2) there are approximately 5,000 to 6,000 new cases each year; 3) current estimates of incidence rates are 120 to 150 cases per 100,000 children <5 years old; 4) KD is 1.5 times more common in males and 85% of cases occur in children <5 years old; and 5) the recurrence rate is low (4%). In 1978, David Morens at the Centers for Disease Control and Prevention published a case definition based on Kawasaki's original criteria. The Centers for Disease Control and Prevention developed a computerized database in 1984, and a passive reporting system currently exists in 22 states. Regional investigations and national surveys suggest an annual incidence of 4 to 15 cases per 100 000 children <5 years o

Child↗
PubMed2003

Platelet glycoprotein IIb/IIIa inhibitors in percutaneous coronary intervention: focus on the pharmacokinetic-pharmacodynamic relationships of eptifibatide.

Eptifibatide is a truncated derivative of the naturally occurring rattlesnake venom protein known as barbourin. It is a cyclic heptapeptide that mimics the tertiary structure found in the parent compound which allows it to bind receptors with the KGD (Lys-Gly-Asp) peptide recognition sequence. Specifically, eptifibatide is a competitive antagonist for the activated platelet glycoprotein IIb/IIIa receptor. Its mechanism of action involves preventing the binding and cross-linking of fibrinogen to the platelet surface. This binding site for fibrinogen is associated with five Ca2+ ions that help maintain the tertiary structure of the receptor and affect the affinity of other ligands such as eptifibatide. Arterial injury induced by percutaneous coronary interventions (PCI) such as balloon angioplasty and stenting, and the spontaneously occurring disease process known as the acute coronary syndrome (ACS), share a common underlying pathophysiology. In both situations, disruption of integrity of the arterial wall initiates a cascade of platelet activation, adhesion and aggregation. Ultimately, this process may proceed to arterial thrombosis unless controlled or modified. Advances in understanding how the platelet plays a pivotal role in this process have significantly enhanced therapy for patients with ACS and have resulted in important reductions in thrombotic complications from PCI procedures. Central to these advances has been evolving understanding of platelet-inhibiting pharmaceutical agents such as eptifibatide. The development of a rational administration regimen for eptifibatide parallels the growth in the understanding of the underlying mechanisms of platelet receptor functions. The binding of eptifibatide to the receptor involves displacement of receptor-associated Ca2+ from the activated binding site. Early in the clinical development of eptifibatide, this was poorly appreciated and resulted in an underestimation of the appropriate doses for this agent. Through a series of small clinical trials and laboratory studies, deficiencies in the early administration regimens were identified and a more effective dose schedule was determined. Modelling of the drug based on its two-compartment pharmacokinetics further defined the role of a newer double-bolus initiation of therapy verses the original single-bolus approach. In a large-scale clinical trial using this double-bolus followed by infusion regimen in PCI procedures, clinical efficacy was shown to be significantly improved over placebo and the earlier, low-dose regimens used in the original trials of eptifibatide.

Angioplasty, Balloon, Coronary↗
PubMed2002

Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity.

A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl-syringyl (G-S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/"p-coumarate-3-hydroxylase" (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H activities. Such enzymes thus fulfill subsidiary processing roles, with all (except CCOMT) apparently being bifunctional for both H and G substrates. Their severe downregulation does, however, predictably result in impaired monolignol biosynthesis, reduced lignin deposition/vascular integrity, (upstream) metabolite build-up and/or shunt pathway metabolism. There was no evidence for an alternative acid/ester O-methyltransferase (AEOMT) being involved in lignin biosynthesis. The G/S lignin pathway networks are operative in specific cell types in angiosperms and employ two additional biosynthetic steps to afford the corresponding S components, i.e. through introduction of an hydroxyl group at C-5 and its subsequent O-methylation. [These enzymes were originally classified as ferulate-5-hydroxylase (F5H) and caffeate O-methyltransferase (COMT), respectively.] As before, neither step has apparently any role in carbon allocation to the pathway; hence their individual downregulation/manipulation, respectively, gives either a G enriched lignin or formation of the well-known S-deficient bm3 "lignin" mutant, with cell walls of impaired vascular integrity. In the latter case, COMT downregulation/mutation apparently results in utilization of the isoelectronic 5-hydroxyconiferyl alcohol species albeit in an unsuccessful attempt to form G-S lignin proper. However, there is apparently no effect on overall G content, thereby indicating that deposition of both G and S moieties in the G/S lignin forming cells are kept spatially, and presumably temporally, fully separate. Downregulation/mutation of further downstream steps in the G/S network [i.e. utilizing 4CL, CCR and CAD isoforms] gives predictable effects in terms of their subsidiary processing roles: while severe downregulation of 4CL gave phenotypes with impaired vascular integrity due to reduced monolignol supply, there was no evidence in support of increased growth and/or enhanced cellulose biosynthesis. CCR and CAD downregulation/mutations also established that a depletion in monolignol supply reduced both lignin contents supply reduced both lignin contents and vascular integrity, with a concomitant shift towards (upstream) metabolite build-up and/or shunting. The extraordinary claims of involvement of surrogate monomers (2-methoxybenzaldehyde, feruloyl tyramine, vanillic acid, etc.) in lignification were fully disproven and put to rest, with the investigators themselves having largely retracted former claims. Furthermore analysis of the well-known bm1 mutation, a presumed CAD disrupted system, apparently revealed that both G and S lignin components were reduced. This seems to imply that there is no monolignol specific dehydrogenase, such as the recently described sinapyl alcohol dehydrogenase (SAD) for sinapyl alcohol formation. Nevertheless, different CAD isoforms of differing homology seem to be operative in different lignifying cell types, thereby giving the G-enriched and G/S-enriched lignin biopolymers, respectively. For the G-lignin forming network, however, the CAD isoform is apparently catalytically less efficient with all three monolignols than that additionally associated with the corresponding G/S lignin forming network(s), which can more efficiently use all three monolignols. However, since CAD does not determine either H, G, or S designation, it again serves in a subsidiary role-albeit using different isoforms for different cell wall developmental and cell wall type responses. The results from this analysis contrasts further with speculations of some early investigators, who had viewed lignin assembly as resulting from non-specific oxidative coupling of monolignols and subsequent random polymerization. At that time, though, the study of the complex biological (biochemical) process of lignin assembly had begun without any of the (bio)chemical tools to either address or answer the questions posed as to how its formation might actually occur. Today, by contrast, there is growing recognition of both sophisticated and differential control of monolignol biosynthetic networks in different cell types, which serve to underscore the fact that complexity of assembly need not be confused any further with random formation. Moreover, this analysis revealed another factor which continues to cloud interpretations of lignin downregulation/mutational analyses, namely the serious technical problems associated with all aspects of lignin characterization, whether for lignin quantification, isolation of lignin-enriched preparations and/or in determining monomeric compositions. For example, in the latter analyses, some 50-90% of the lignin components still cannot be detected using current methodologies, e.g. by thioacidolysis cleavage and nitrobenzene oxidative cleavage. This deficiency in lignin characterization thus represents one of the major hurdles remaining in delineating how lignin assembly (in distinct cell types) and their configuration actually occurs.

Gene Expression Regulation, Plant↗
PubMed1997

Differences among patients in opioid self-administration during bone marrow transplantation.

The distinctive features of individual patients, here termed individual differences, are inescapable aspects of day-to-day patient pain management, but classically designed research studies ignore such differences. This paper introduces statistical pattern visualization methodology for the study of complex individual differences in clinical settings. We demonstrate the application of such methods in patients undergoing bone marrow transplantation (BMT) and suffering severe oral mucositis as a consequence of the aggressive BMT preparative regimen. Oral mucositis produces severe pain and patients often require parenteral opioid medication for several weeks. Unfortunately, the opioid can cause side-effects that limit drug use for pain control. Patients differ in severity and duration of oral mucositis, analgesic response to opioids, and side-effects. We identified and classified individual differences in patterns of drug use, pain control and side-effects in 33 BMT patients who received opioid drug via patient-controlled analgesia (PCA) systems for 7 days or more. These systems allowed bolus dosing and also provided a basic level of analgesic protection through continuous drug infusion. Continuous infusion levels increased or decreased in response to patient bolus self-administration. We employed statistical smoothing (moving average) techniques to remove random variation from the individual data sets and created three-way (trivariate) plots of change over time in drug use, pain and an opioid side-effect (impairment of concentration). The patterns apparent in these plots indicated that 24.2% of patients used PCA optimally (increases in drug use associated with reductions in pain and little or no side-effect), an additional 30.3% manifested a potentially optimal pattern limited by side-effect that worsened with dosing, and 36.4% used PCA suboptimally (modest pain control plus side-effects). In addition, for each subject we created a summary measure for the simultaneous change in three variables: the distance of each day's trivariate score from the origin of a three dimensional plot. This summary measure correlated significantly with the changing severity of patients' oral mucositis over time (r = 0.502). This study demonstrates how interactive graphic techniques can provide a basis for examining changes over time among multiple, correlated variables associated with a single individual. It illustrates the application of such techniques and demonstrates that individual subject data sets merit examination in cases where clinical data reflect human performance.

Adolescent↗
PubMed1998

Basics of flow cytometry.

In summary, a beginner requires fundamental knowledge about flow cytometric instrumentation in order to effectively use this technology. It is important to remember that flow cytometers are very complex instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acquired data and is also the user interface for controlling most instrument functions. These four systems provide a very unique and powerful analytical tool for researchers and clinicians. This is because they analyze the properties of individual particles, and thousands of particles can be analyzed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement. Basic knowledge of instrumentation is a tremendous aid to designing experiments that can be successfully analyzed using flow cytometry. For example, it is important to know the emission wavelength of the laser in the instrument that will be used for analysis. This wavelength is critical knowledge for selecting probes. It is also important to understand that a different range of wavelengths is detected for each fluorescent channel. This will aid selection of probes that are compatible with the flow cytometer. Understanding the complication that emission spectra overlap contributes to detection can be used to guide fluorochrome selections for multicolor analysis. All of these experiment design considerations that rely on knowledge of how flow cytometers work are a very practical and effective means of avoiding wasted time, energy, and costly reagents. Data analysis is a paramount issue in flow cytometry. Analysis includes interpreting as well as presenting data that has been stored in list-mode files. Data analysis is very graphically oriented. There are a number of types of graphic representation that are available to visually aid data analysis. Two standard types of displays are used. These data plots are one-parameter histograms and bivariate plots. A user must be familiar with these two fundamental types of display in order to effectively analyze data. Histograms are the most simple modes of data representation. Histograms allow visualization of a single acquired parameter. Mean fluorescence and distributional statistics can be obtained based on markers that the user can graphically set on the plot. Percentages of positively expressing particles relative to a control sample can also obtained in a similar manner. In addition, multiple histograms can be overlayed on one another to depict qualitative and quantitative differences in two or more samples. Two-parameter data plots are somewhat more complicated than histograms; however, they can yield more information. Two-parameter dot plots of FSC vs SSC allow visualization of both light-scattering parameters that are important for identifying populations of interest. Bivariate fluorescent plots allow discrimination of dual-labeled populations that might remain hidden if histograms were used to display fluorescent data. Two-parameter plots that combine one light-scattering parameter and a fluorescent parameter are useful for analyzing control samples to elucidate the origin of nonspecific binding. Data analysis is very graphically oriented. Experience and pattern recognition become important when using two-parameter data plots for qualitative as well as quantitative analysis. The technique of gating or drawing regions on dual parameter light-scatter plots allows one to exclude information and examine the population of interest by disallowing particles that might confound or interfere with analysis. This is one of the fundamental uses for gating. (ABSTRACT TRUNCATED)

Animals↗
PubMed1998

Specificity of humoral and cellular immune response against recombinant particles of nucleocapsid protein of human hepatitis B virus in rabbits.

Nucleocapsid (core) protein of hepatitis B virus (HBcAg) induces potent cellular and humoral responses that have a clear protective potential. Rabbits were immunized by particles formed by recombinant molecules of HBcAg carrying N-terminally inserted heterologous sequences. Specificity of humoral and cellular immune response against HBcAg and selection of HBcAg epitopes was surveyed. Immunological properties of the recombinant particles were similar to those of the original HBcAg. Recombinant particles were not toxic to the peripheral blood mononuclear cells (PBMC) of non-immune or HBcAg-immunized animals ex vivo. Proliferative response of PBMC (T-lymphocytes) to HBcAg in immunized animals increased in a concentration-dependent manner in the broad interval of HBcAg concentrations (10-104 ng/ml). On the contrary, a narrow bell-shaped HBcAg dose-dependence curve was earlier observed for T-lymphocytes of donors immune to HBV after natural infection that was probably due to the cytotoxic effect of HBcAg on the expressing cells. Specificity of humoral and cellular immune response against HBcAg particles in the immunized animals and in natural infection with hepatitis B virus (HBV) was compared. Immunization with recombinant HBcAg particles induced potent anti-HBcAg antibody responses: high (up to 2.107) titers of anti-HBcAg antibodies were reached. Appearance of anti-HBcAg antibodies was in every case preceded by an increasing T-cell response to the whole protein and HBcAg-derived peptides, thus mimicking immune responses during acute HBV infection in humans. A predominant universal (haplotype-independent) T-helper cell epitope (amino acid residues (aa) 61-85 of HBcAg (p61-85)) was recognized by T-cells of all animals. Transient antibody response against p61-85 was recorded during the early stages of immunization in spite of the fact that a major B-cell epitope localized in this region is supposed to be purely conformational. A sequence representing another cluster of immunodominant T-cell epitopes of mice and HBV infected humans, aa 121-140 (p121-140), was not immunogenic on the T-cell level. However, it appeared to be a potent B-cell immunogen, despite a common assumption that HBcAg and p121-140 are not cross-reactive at the B-cell level. A possibility that anti-p121-140 antibodies were induced by an exposed region of the native particulate HBcAg and not by the denatured protein molecules, was confirmed by recognition of the particulate HBcAg by antibodies specific to synthetic peptides representing aa 120-140 of HBcAg. The data point to the exposition of aa 121-140 on the surface of the particles.

Amino Acid Sequence↗
PubMed2004

Modern diagnostics in chronic myeloproliferative diseases (CMPDs).

According to the new WHO classification a group of chronic myeloproliferative diseases (CMPDs) were defined: chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia and hypereosinophilic syndrome (CEL/HES), polycythemia vera (PV), chronic idiopathic myelofibrosis (with extramedullary hematopoiesis, CIMF), essential thrombocythemia (ET), and so called CMPD/unclassifiable. As clinical features and laboratory findings differ widely between these diseases several diagnostic approaches are mandatory at diagnosis for classification and are needed also for follow up studies, especially for the measurement of minimal residual disease (MRD). We here outline the laboratory set up at diagnosis and during follow up in CMPDs with specific focus on the respective therapeutical consequences. Only by using a comprehensive diagnostic panel including cytomorphology, cytogenetics, and molecular genetic methods establishing the correct diagnosis, optimizing treatment as well as evaluating treatment response is possible in CMPDs today.

Blood Cell Count↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗