Nonspecific Events Research Articles

Primary developmental field. III: Clinical and epidemiological study of blastogenetic anomalies and their relationship to different MCA patterns.

Opitz [1993: BD:OAS XXIX (1):3-37] suggested that during blastogenesis the entire embryo constitutes a developmental field, i.e., the primary developmental field. Based on this principle, he postulated that a single "hit," that during late morphogenesis would cause a monotopic malformation, during blastogenesis would produce a polytopic malformation or an association. Lubinsky [1986: Am J Med Genet [Supp1]2:6-16] had stated previously that "since the embryo develops in an integrated manner, organized and differentiating spatially, temporally, and in an epimorphically hierarchical manner, disturbances result in nonrandom patterns of anomalies." He then concluded that "associations are derivatives of causally nonspecific disruptive events acting on developmental fields." These concepts, confirmed by our epidemiological observations [Martínez-Frías, 1994: Am J Med Genet 49:45-51], imply that some associations are, by definition, abnormalities of blastogenesis that is, that their component congenital anomalies are produced by events occurring during the first 4 weeks of development. We present an analysis of the characteristics of blastogenetic anom-alies and their relationship with midline abnormalities, as well as with the schisis and VACTERL associations.

Biochemistry of demyelination.

The myelin sheath, a lipid-rich multilamellar membrane of relative stability, both insulates and enhances conduction in nerve axons. A notable feature of myelin-specific proteins, in particular myelin basic protein, is their susceptibility to proteolytic activity and their encephalitogenicity, which induces inflammatory demyelination in the CNS. The final common pathway of myelin breakdown in vivo is well documented and there is evidence that myelin disruption can be mediated directly by soluble (circulating) factors and for following receptor-driven phagocytosis by macrophages. However the exact mechanism(s) of demyelination in multiple sclerosis is still unresolved, both antigen-specific and--non-specific events having the potential to generate the myelinolytic process.

CURRENT CONCEPTS OF THE ETIOLOGY AND PATHOGENESIS OF ULCERATIVE COLITIS AND CROHN’S DISEASE

Although the causes, events initiating and triggering inflammation, and the precise immunoregulatory defects of IBD are still not known, investigations have provided a better understanding of the mechanisms of perpetuation of inflammation, genetic susceptibility, tissue injury, and symptoms. Ulcerative colitis and Crohn's disease are related disorders that probably share susceptibility genes and have similar nonspecific inflammatory mediator profiles. These diseases, however, almost certainly have different causes and respond to different antigenic stimuli. It is probable that both ulcerative colitis and Crohn's disease represent heterogenic groups of diseases that share similar mechanisms of tissue damage but have different initiating events and immunoregulatory abnormalities. Rodent models demonstrate that a wide variety of initial injuries or perturbations of immunoregulatory pathways can lead to similar phenotypes of intestinal injury, and human studies show evidence of genetic heterogeneity. It is equally apparent from these models that initiating and perpetuating mechanisms are entirely distinct and that the intestine has a remarkable ability to heal. Chronicity of disease depends on continued exposure to toxic luminal components, most commonly of bacterial origin, and genetically determined host susceptibility. Precise mechanisms of differential genetic susceptibility remain unclear, but defective down-regulation of inflammation is consistent with clinical and experimental observations. The author proposes the following sequence of events (Fig. 9). Nonspecific intestinal inflammation can be induced by a wide variety of enteric infections or ingested toxins. Resultant increased mucosal permeability leads to enhanced uptake of toxic luminal bacterial products, which potentiate local injury. The vast majority of hosts respond to these injurious events by promptly down-regulating the inflammatory response and rapidly healing the mucosal damage without residual scarring. The genetically susceptible host, however, who lacks the ability to suppress the inflammatory response efficiently, has inappropriate amplification of the immune cascade. In response to constant exposure to phlogistic luminal constituents, these patients develop an unrestrained inflammatory response, leading to tissue destruction, chronic inflammation, and fibrosis. Thus, IBD is caused by a genetically determined defective down-regulation of inflammation driven by ubiquitous antigens. Luminal anaerobic bacterial antigens are the stimuli in Crohn's disease, but ulcerative colitis may be caused by functionally abnormal aerobic bacteria or primary defects in epithelial cell physiology. Spontaneous or therapy-induced remissions can be achieved, but the risk of reactivation of inflammation is high because of the frequent exposure to triggering episodes that can reignite the inflammatory cascade. [formula: see text] This theory suggests that the intestine is in a constant state of controlled inflammation, mediated by a balance between aggressive luminal forces and host protective mechanisms (Fig. 10). This delicate balance can be deranged by any number of environmental triggering events and is in dysequilibrium in IBD. Amplification of the inflammatory response activates effector cells and cascades of soluble inflammatory molecules, which mediate tissue injury and physiologic responses leading to symptoms of IBD. These relatively nonspecific events are the target of most current therapeutic agents, which can inhibit but not completely block intestinal inflammation because of the overwhelming number of parallel pathways involved. Specific inhibition of selected effector molecules is intellectually intriguing but is less likely to paralyze the inflammatory response during clinically apparent inflammation than is blockade of key immunoregulatory cells and molecules. Better understanding of initiating, perpetuating, and immunoregulatory mechanisms should provide more

An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry

The use of the chromium-release assay to determine cytotoxicity of effector against target cells has various limitations mostly due to the inherent properties of the radioactive substance. We have developed an improved flow cytometric method that is able to measure cytotoxicity, based on two fluorescent dyes. Calcein-AM, a non-fluorescent substance which is intracellularly converted to the green fluorescent calcein by esterase activity in viable cells, is initally used to stain target cells. After incubating targets with effectors for 2 h, ethidium homodiner-1,a red DNA stain non-permeable to viable cells, is added. Dead target cells are distinguished by their double (green-red) staining. Data analysis is performed by gating the regions of living target, dead target and living effector cells, based on appropriate controls. Non-specific events are subtracted from the dead target region and the ratio of specific dead target events to total target events is expressed as percent cytotoxicity. The method is used to quantify natural killer (NK) and lymphokine-activated killer (LAK) activities against the human K562 and Daudi cell lines and the murine YAC-1 and L1210 cell lines respectively, as well as cell-mediated lympholysis (CML) exerted by tumor-infiltrating lymphocytes (TIL) against autologous and allogeneic human breast cancer tumor cells. The method is fast, reliable and correlates well with the standard 51Cr-release assay.

Developmental field defects and associations: epidemiological evidence of their relationship.

Lubinsky [1986: Am J Med Genet 2:9-16] has defined associations as derivatives of causally nonspecific disruptive events acting on developmental fields. Opitz [1992: Second International Workshop on Fetal Genetic Pathology] considers developmental fields as the basic biologic units of individual development and of evolution, and has stated that associations represent the idiopathic occurrence of multiple congenital anomalies during blastogenesis. These concepts imply that associations represent the concurrence of a greater number of developmental field defects (DFDs) than other patterns of multiple anomalies. The coding system for children with multiple congenital anomalies, developed in the Spanish Collaborative Study of Congenital Malformations (ECEMC), permits analysis of DFDs as morphogenetic units. Thus, we can study their presence in any type of MCA pattern, regardless of its etiology. Here we present the analysis of a selected group of DFDs of blastogenic origin. Specifically, we have studied how the groups of defects, usually comprised in those specifics DFDs, are observed in children who present different clinical entities such as associations, embryopathies, syndromes of known etiology, and others. The results of our analysis show a greater concurrence of the selected DFDs in associations than in other MCA patterns, and support the concept of Lubinsky [1985: Am J Med Genet 21:35-38; 1986] and Opitz [1992].

Functional Disturbances of the Autonomic Nerve in Nasal Hyperreactivity: An Up-date Review

During the 1980s, our view of airway hypersensitivity was altered significantly. Advances in biochemical techniques revealed involvement of several nonspecific events in nasal hyperreactivity: Autonomic dysfunction involving primary and/or secondary receptor disorders, epithelial damage by cytotoxic proteins in eosinophil, which is stimulated by inflammatory mediators, and an axonal reflex of sensory C fibers. Since 1983, we have neurobiochemically investigated the autonomic nerve dysfunction in the nasal mucosa of patients with nasal allergy and guinea pigs with experimentally-induced nasal hypersensitivity. We propose the following mechanisms as potential contributors to the disturbance of the beta receptor function in airway hyperreactivity: i) Down-regulation caused by excess endogenous norepinephrine stimulation, ii) down-regulation and uncoupling to adenylate cyclase, produced by the inflammatory mediator-induced activation of protein kinase C, iii) the action of beta receptor inhibitory factor, presumably anti beta receptor autoantibodies, and iv) dysfunction of beta receptor kinase, which is known to cause short-term desensitization of beta receptors after exposure to beta agonists. This review provides the anatomical and neurobiochemical background for the autonomic regulation and dysfunction in the nose. We also introduce our series of experiments and the above updated hypotheses of how functional disturbances of the autonomic nerve in the nasal mucosa may occur.

Origins of DNA replication in metazoan chromosomes.

In metazoan chromosomes, initiation of DNA replication occurs primarily at specific sites (0.5-3 kb; the OBR) using the same replication fork mechanism favored by simple genomes. Nucleosome segregation is distributive. However, initiation events also occur at other sites randomly distributed throughout a larger initiation zone (8-55 kb). These nonspecific initiation events presumably occur at a lower frequency than those at the OBR. Although origins in native chromosomes appear to be genetically determined, ori sequences have been convincingly demonstrated only during programmed gene amplification. It seems likely that metazoan origins include both specific DNA sequences as well as chromatin structure and nuclear organization, something that may be difficult to reproduce with plasmid DNA. Definitive answers will come only with a functional assay for initiation that exhibits the sequence-specific characteristics observed in vivo.

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

Effects of T Antigen and Replication Protein A on the Initiation of DNA Synthesis by DNA Polymerase α-Primase

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

The Clinical Safety of Omeprazole

This review comprises data from more than 19,000 individuals who have taken part in clinical studies of omeprazole. Isolated, non-specific adverse events which might be attributable to omeprazole have included nausea, dizziness, headache and diarrhoea. These events have been generally mild and transient and have not usually required either a reduction of dose or cessation of therapy. The frequency and spectrum of adverse events have been the same in those over 65 years of age as in younger patients. No drug-related adverse events have been found in patients with renal insufficiency or severe liver failure. More than 1.2 million patient treatments of omeprazole have now been given. The overall incidence of adverse events with omeprazole is low, and in comparative studies has been in the same range as that found with H2-receptor antagonists. Importantly, no dose-related adverse events have been observed with omeprazole in the dose range 10-60 mg/day. Furthermore, none of the serious adverse events that have been reported have been attributable to omeprazole. No histological changes in oxyntic endocrine cells have been found after short-term periods of treatment with either omeprazole or H2-receptor antagonists in patients with peptic ulcer disease. Long-term continuous high-dose omeprazole treatment of patients with Zollinger-Ellison syndrome has not induced any significant increase in the oxyntic endocrine cell hyperplasia. Investigations of the gastric mucosa from patients in a compassionate use programme who have received omeprazole, usually 20 mg daily, for periods of up to 37 months, have been performed. Two hundred and forty-eight patients had their last biopsy taken after at least 11 months of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Humoral and cellular mechanisms in acute allograft injury

An intact thymus-competent immune system seems both necessary and sufficient for acute allograft rejection. T-lymphocytes fulfill a form of Kochs postulate in this regard: (1) They are present in rejecting allografts; and (2) rejection is abrogated by depriving the host of T cells but can be recreated by providing such T cell-deficient hosts with purified T cells. However, the dilemma of whether Th or Tc cells actually mediate allograft rejection remains. Although belonging to opposite immunologic "teams," they act on each other in many ways, interactions that are probably just the beginning of the complex cascade of both specific and nonspecific host immunologic events, both cell mediated and humoral, that are stimulated by the presence of allogeneic tissues.

Acute sensitivity of BHT-induced alveolar toxicity to a diquat challenge in murine lungs

In this study we investigated the effects of a mild toxic challenge at selected points in time on the nonspecific cellular events that occurred in acutely damaged pulmonary alveoli. Swiss-Webster mice were treated with butylated hydroxytoluene (BHT, 400 mg/kg ip) and sacrificed at Days 1, 3, and 5 thereafter; either 24 or 48 hr prior to each sacrifice, the herbicide diquat was administered (4 mg/kg ip) as a challenge to the ongoing cellular events in the pulmonary alveoli. Standard morphometric techniques were used at both the levels of light and electron microscopy to evaluate the alveolar response to BHT and diquat treatments. Following BHT, early type I epithelial and endothelial damage triggered inflammatory changes in alveolar septa. Proliferation and differentiation of type II pneumocytes, aiming at the regeneration of the respiratory epithelium, ensued and peaked at Day 3. Treatment with diquat alone caused mild inflammatory changes and hypertrophy of type II pneumocytes, in the absence of necrosis of any alveolar cell type. The pinpoint administration of diquat in the early days following treatment with BHT significantly disorganized the temporal pattern of alveolar reaction. Diquat enhanced epithelial and endothelial damage only if administered before the onset of BHT-induced injury. Afterwards, the alveolar response to the combined effects of BHT and diquat could not be predicted from their known individual effects. Treatment with diquat modified either proliferation or differentiation of type II pneumocytes, depending upon time along the BHT schedule. Inflammatory and interstitial reactions were lowered when diquat was given at Days 1 and 3 post-BHT, but potentiated when given at Day 4. The results document time-related changes in the sensitivity of damaged and regenerating alveolar cells to a mild exogeneous chemical challenge. They may further indicate that low levels of urban and industrial toxicants might influence pulmonary alveolar events in individuals made more susceptible by acute or chronic respiratory diseases.

Perforated ulcers in critical illness.

Although gastroduodenal ulcers rarely perforate during critical illness, this occurrence causes specific and difficult problems of diagnosis and management. In our review of the records of 19 critically ill patients whose ulcers perforated, we found that classical symptoms were frequently absent. The perforations often were manifested by nonspecific clinical events, such as unexplained ileus or hypotension. Diagnosis was typically delayed and on occasion was first suspected after observing pneumoperitoneum on a routine x-ray. While the ulcers were characteristically very large (greater than 2 cm), minimal inflammation surrounded them. Mortality was 56% in the 16 patients whose perforations were diagnosed before death or discharge. Fifteen patients were treated with simple patching of the ulcer. Perforated ulcers in critically ill patients differ in several important ways from those occurring in otherwise healthy individuals.

VATER and other associations: historical perspectives and modern interpretations.

Associations are statistically related groups of anomalies, and the VATER association probably is the best known example. The concept is widely used, but there are difficulties with both definition and delineation. An historical review is used to define implicit commonalities of associations. These commonalities are then used to develop a testable model which defines associations as derivatives of nonspecific teratologic events acting on developmental fields. Findings depend on the timing, location, and magnitude of these events, and reflect developmental constraints. This clarifies the nature of associations and provides guidelines for their study and use.

Therapy credibility is not a nonspecific event

Therapy credibility and client outcome expectancies instilled by psychotherapy procedures have been classified as nonspecific factors. Their control has been required in psychotherapy research, just as nonspecific factors must be controlled in chemotherapy research. However, an examination of the definitions of “nonspecific” shows that credibility and instilled expectancies do not qualify as nonspecific events. To maintain that they are nonspecific factors requires a distortion of the meaning of nonspecific. In psychotherapy research, it is not necessary to control credibility in the name of controlling nonspecific artifacts. Implications are presented for psychotherapy research, theory, and language.

Initial characterisation of the sheep immune response to infections of Lucilia cuprina

Assays for total serum antibody, histamine sensitivity and the presence of reaginic antibodies were carried out on sheep repeatedly infected with first stage larvae of Lucilia cuprina. Effects of the sheep response on the larvae were monitored at the final infection and compared with control animals by the recovery of larvae and measurement of the wound caused by the larvae. Overall larval survival was not significantly different in the pre-infected group though wound sizes were smaller. Histamine sensitivity appeared to correlate with wound size only in the control group. Thus recent infection experience may lead to immune responses which override non-specific inflammatory events and cause smaller wound sizes. A sub-group of the pre-infected sheep had lower larval survival and smaller wound sizes than the other animals and this correlated with increased levels of reaginic antibody and lower total antibody levels. The results suggest a genetic basis for resistance to fly strike and the possible involvement of reaginic antibody in protective responses.

Cellular basis of allograft rejection.

In summary, we currently hypothesize that systemic immunization within the lymphoid tissue is amplified by a sequence of specific and non-specific immunological interactions which lead to enrichment of specifically sensitized T-cells at the graft site. Nascent sensitized cells enter the graft via non-specific attractants generated by the healing response. The interaction of alloantigen with responder T-cells bearing specific alloantigen receptor results in production of lymphokines which increase blood flow and vascular permeability, and which are chemotactic for circulating lymphocytes. These events and other non-specific events recruit circulating cells regardless of the specificity of their sensitization. The state of activation of these cells may control their presence within the circulation. Cells which are more activated are released more readily from the lymphoid depots into the circulation so that relatively more specifically sensitized (though not necessarily mature cytotoxic cells) cells would enter the circulation and be recruited to the graft. At the allograft, there is an escalating sequence of lymphokine production and non-specific recruitment. II-1 has been demonstrated to be capable of mediating such recruitment. A second phase of amplication occurs at the graft site where precytotoxic (x-ray-sensitive) cells mature and expand into cytotoxic (x-ray-resistant) cells. These cells may be important effector cells of allograft rejection in concert with many unsensitized cells which can participate in non-specific attacks on graft viability. The essential point made from the studies summarized here is the consideration that trafficking patterns of lymphocytes and recruitment of lymphocytes and other inflammatory cells are in essential component to the local mechanism of graft rejection. The relative importance of specificity of response has not been defined.

Programming of DNA rearrangements involving mu prophages.

Most of the papers in this Symposium provide abundant evidence that changes in DNA sequence organization — and, consequently, changes in the information content of DNA molecules — result from the action of specific biochemical systems. Over the past three decades, detailed in vivo and in vitro studies of how genetic changes occur have radically altered our ideas about the mutational process. Instead of invoking nonspecific chemical events, such as keto-enol tautomerism in purine and pyrimidine bases, we now see specific enzymatic activities as the agents of genetic change. Systems like the SOS response of enteric bacteria (Witkin 1976) show that genetic change is a biochemical process even when there has been direct physicochemical damage to DNA molecules. Some kinds of DNA reorganization, such as homology-dependent general recombination and replicative recombination, involve multiple biochemical steps and the activities of many proteins. There is, therefore, prima facie evidence for regulatory processes...

Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria.

A higher molecular weight precursor (Mr = 39,000) to the liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = 36,000), is imported and processed by heart mitochondria in vitro in a manner similar to liver mitochondria. In both systems, however, an additional 37-kDa ornithine carbamyltransferase polypeptide appears, but this arises from nonspecific events and, therefore, does not represent a bona fide intermediate in the overall processing sequence. Our experiments demonstrate that the outer mitochondrial membrane of mitochondria contains a protease-sensitive (5 micrograms of trypsin or chymotrypsin/ml, 15 min at 2 degrees C), salt-resistant (1.0 M KCl) protein which is required to maintain import functions. In addition, functional post-translational import requires a component of the reticulocyte lysate (i.e. cytosol) that is used for initially synthesizing precursor enzyme. The component is retained by Sephadex G-25. Import of Sephadex G-25-excluded precursor is restored by fresh reticulocyte lysate but not by a combination of other additives, including Mg2+, K+, ATP, ADP, Pi, succinate, and total translation mixture (minus lysate).

Cholinesterase activities in the somatic nervous system of rabbits with experimental allergic neuritis

The allergic inflammatory disorders of the nervous tissue are associated with a complex series of cellular and humoral immune activities and they usually result at least in demyelination, but according to morphologic evidence also in secondary neuronal changes. Using the colorimetric method of Ellman et al. (G. L. Ellman, K. D. Courtney, V. Anders, and R. M. Featherstone, 1961, Biochem. Pharmacol. 7: 88–95) the activities of enzymes splitting acetylthiocholine iodide (AThCh) were determined from various parts of the somatic nervous system of rabbits with experimental allergic neuritis (EAN), a primary demyelinating disease of the peripheral nerves. It was found that the total activity of AThCh-splitting enzymes was decreased already in an early phase of the disease in the dorsal root ganglia (DRG). In a well developed phase of the disease the activity of acetylcholinesterase (AChE) seemed to be decreased by 33% in the ventral roots and by a lesser amount in the DRG and the most proximal part of the sciatic nerves. The mechanism of the recorded changes may be related either to specific or to nonspecific immune events or to both. Proteolytic activity released by macrophages in the target tissue may, by inactivating the hydrolytic activity of AChE, at least partly explain these findings. Because the activity of AChE in the structures studied derives from a neuronal origin, our results provide biochemical evidence for the involvement of neurons in the sensory ganglia and of axolemma in the ventral roots in EAN.