A 7-day intravenous toxicity study and neurotoxicity assessment of pyridorin in Sprague-Dawley rats.

Pyridorin®, a naturally occurring metabolite of vitamin B6 that inhibits and scavenges reactive oxygen species, is being developed as a potential therapeutic for acute kidney injury. An investigational new drug application (IND) was opened for Pyridorin in support of its ongoing oral drug clinical development program. Currently, a Pyridorin intravenous (IV) formulation is being developed for use in surgical patients. To support the IND for Pyridorin, a full battery of nonclinical Good Laboratory Practice compliant studies was performed with no neurological or behavioral signs of toxicity seen following oral or IV administration of pyridoxine dihydrochloride (the active ingredient in Pyridorin). However, excessive ingestion of vitamin B6 has been reported to cause neurotoxic syndrome in humans. Therefore, under Food and Drug Administration recommendation, a 7-day IV study in rats was conducted to further evaluate the drug's potential to cause neurotoxicity. Blood plasma samples indicated that exposure to pyridoxamine dihydrochloride and its metabolites, pyridoxal, pyridoxine, and 4-pyridoxic acid was linearly dose proportional and independent of gender. At doses of up to 200 mg/kg/day pyridoxine dihydrochloride, no treatment-related effects were seen in rats, providing further evidence for the absence of pyridoxine dihydrochloride-related changes in the nervous system. A no observed adverse effect level of 200 mg/kg/day was identified for this study.

The formation of neural codes in the hippocampus: trace conditioning as a prototypical paradigm for studying the random recoding hypothesis.

The trace version of classical conditioning is used as a prototypical hippocampal-dependent task to study the recoding sequence prediction theory of hippocampal function. This theory conjectures that the hippocampus is a random recoder of sequences and that, once formed, the neuronal codes are suitable for prediction. As such, a trace conditioning paradigm, which requires a timely prediction, seems by far the simplest of the behaviorally-relevant paradigms for studying hippocampal recoding. Parameters that affect the formation of these random codes include the temporal aspects of the behavioral/cognitive paradigm and certain basic characteristics of hippocampal region CA3 anatomy and physiology such as connectivity and activity. Here we describe some of the dynamics of code formation and describe how biological and paradigmatic parameters affect the neural codes that are formed. In addition to a backward cascade of coding neurons, we point out, for the first time, a higher-order dynamic growing out of the backward cascade-a particular forward and backward stabilization of codes as training progresses. We also observe that there is a performance compromise involved in the setting of activity levels due to the existence of three behavioral failure modes. Each of these behavioral failure modes exists in the computational model and, presumably, natural selection produced the compromise performance observed by psychologists. Thus, examining the parametric sensitivities of the codes and their dynamic formation gives insight into the constraints on natural computation and into the computational compromises ensuing from these constraints.

Quantal synaptic failures enhance performance in a minimal hippocampal model.

Despite the fact that animals are not optimal, natural selection is an optimizing process that can readily control small bits and pieces of organisms. It is for this reason that we need to explain certain parameters as found in Nature (e.g., number of neurons and their average activity) to fully understand the biological basis of cognition. In this optimizing sense, the failure of quantal synaptic transmission is problematic because this process incurs information loss at each synapse which seems like a bad thing for information processing. However, recent work based on an information-theoretic analysis of a single neuron suggests that such losses can be tolerated and lead to energy savings. Here we study computational simulations of a hippocampal model as a function of failure rate. We find that the failure process actually enhances some indices of performance when the model is required to solve the hippocampally dependent task of transverse patterning or when it is required to learn a simple sequence. Adding the random process of synaptic failures to the recurrent CA3-to-CA3 excitatory connections results in simulations that are more robust to parametric settings. Not only is the model more robust when synaptic failures are part of the model but there is a notable increase of sequence length memory capacity. Also, the failure process combined with additional neurons allows lower activity settings while still remaining compatible with learning the transverse patterning task. Indeed, as neuron number tended towards the biological numbers (nearly 5 x 10(4) in the simulations), it was not only possible to achieve biological failure rates (55-85%) at the minimally tolerated activity setting but these appropriately high failure rates were required for successful learning. The results are interpreted in terms of previous research demonstrating that randomization during training can enhance performance by facilitating implicit state-space search for interconnected neurons.

Initial state randomness improves sequence learning in a model hippocampal network.

Randomness can be a useful component of computation. Using a computationally minimal, but still biologically based model of the hippocampus, we evaluate the effects of initial state randomization on learning a cognitive problem that requires this brain structure. Greater randomness of initial states leads to more robust performance in simulations of the cognitive task called transverse patterning, a context-dependent discrimination task that we code as a sequence prediction problem. At the conclusion of training, greater initial randomness during training trials also correlates with increased, repetitive firing of select individual neurons, previously named local context neurons. In essence, such repetitively firing neurons recognize subsequences, and previously their presence has been correlated with solving the transverse patterning problem. A more detailed analysis of the simulations across training trials reveals more about initial state randomization. The beneficial effects of initial state randomization derive from enhanced variation, across training trials, of the sequential states of a network. This greater variation is not uniformly present during training; it is largely restricted to the beginning of training and when novel sequences are introduced. Little such variation occurs after extensive or even moderate amounts of training. We explain why variation is high early in training, but not later. This automatic modulation of the initial-state-driven random variation through state space is reminiscent of simulated annealing where modulated randomization encourages a selectively broad search through state space. In contrast to an annealing schedule, the selective occurrence of such a random search here is an emergent property, and the critical randomization occurs during training rather than testing.

Immunization with recombinant Pneumocystis carinii p55 antigen provides partial protection against infection: characterization of epitope recognition associated with immunization.

Many therapeutic options exist for the treatment of Pneumocystis carinii pneumonia, a common fungal opportunistic pulmonary pathogen, but treatment is often complicated by side effects and toxicity and, more recently, markers of drug resistance have been described. The development of immunotherapetic modalities such as active immunization or passive immunotherapy may play an increasing important role in the prevention and treatment of infection. Passive immunotherapy with polyclonal anti-P. carinii reagents, such as serum or T cells, and monospecific reagents reactive with the major surface glycoprotein (MSG or gpA), such as monoclonal antibodies or MSG primed T cells, reduce the severity or eradicate infection. Active immunization with whole P. carinii, P. carinii extracts or MSG has afforded partial protection against the subsequent development of P. carinii pneumonia in some animal models. Identification of additional antigens with protective benefits will aid in the development of vaccines or other reagents. The p55 antigen of rat-derived P. carinii is well recognized by animals following natural exposure to the organism. This 414 amino acid residue antigen found within the cell wall of P. carinii contains 7 repeats of a glutamic acid-rich motif in the carboxyl portion of the molecule. Both humoral and cellular immune responses reactive with this repeated domain are present following natural infection while, the amino terminal portion of the molecule is immunologically silent. In this study, immunization with recombinant p55 elicited significant humoral and cellular immune responses which persisted during 10 weeks of immunosupression in corticosteroid treated rats; rp55 immunization resulted in a significant reduction in organism burden, improved histological score, lower lung weight to body weight ratio (a marker of infection or lung inflammation) and improved survival (P < 0.01). Greater protection was afforded by immunization with a peptide containing amino acid residues 1-200, than by the entire rp55 molecule. Epitope recognition by serum from animals immunized with rp55 differed from that of naturally exposed animals with oligoclonal responses to residues 22-92 and residues 196-218. This study demonstrates that protection against P. carinii can be afforded by immunization with antigen preparations other than whole extracts of P. carinii or the major surface antigen, MSG. This antigen moiety will likely be most useful as a vaccine candidate in combination with other immunogens which provide similar partial protection.

Cytokine responses to the native and recombinant forms of the major surface glycoprotein of Pneumocystis carinii.

Pneumocystis carinii is a major opportunistic pathogen and leading cause of morbidity in patients with AIDS. The major surface glycoprotein (MSG) of P. carinii, represented by a family of related proteins encoded by unique genes, is highly immunogenic and contains T cell-protective epitopes. We undertook the present study to define the CD4 T helper (Th) response by cytokine secretion to native MSG and a recombinant form of the protein, MSG-B. Spleen cells were collected from Lewis rats and restimulated with both native MSG and MSG-B. Within 24 h, the CD4 cells secreted high levels of interferon-gamma (IFN-gamma) in response to both types of antigen, indicative of a Th1 response; however, after 72h of incubation, only the native MSG stimulated secretion of IL-4 (Th2 response) from the cells. We then investigated whether the presence of IL-4 could alter the predominant Th1 phenotype by the CD4 cells in response to MSG and MSG-B. Cells cultured with native MSG and IL-4 produced low levels of IFN-gamma and elevated levels of IL-4. Interestingly, cells incubated with MSG-B and IL-4 reduced production of IFN-gamma, but were not stimulated to produce increased levels of IL-4. The presence of anti-IFN-gamma antibody in the MSG- or MSG-B-stimulated cultures did not effect the expression of IFN-gamma mRNA, suggesting that the generation of Th1 cells in response to MSG or MSG-B was not dependent on IFN-gamma. We conclude that native MSG, which contains multiple forms of this antigen, and recombinant MSG elicit different cytokine responses in vitro. These data are not only important to studies of MSG, but may also be relevant to the role of MSG in the immunopathogenesis of P. carinii infection in vivo.

Cellular responses to a 55-kilodalton recombinant Pneumocystis carinii antigen.

The host-parasite interaction in Pneumocystis carinii pneumonia is poorly understood. In recent years, two major groups of P. carinii antigens have been identified. One class of antigens is characterized by a broad band of immunoreactivity between 45 and 55 kDa in P. carinii derived from rats. This antigen complex is the P. carinii antigen most commonly found in respiratory tract specimens and most frequently recognized by the host immune response. The availability of a recombinant antigen has permitted studies focusing on the cellular and humoral responses to a single antigen within this class, p55. In this study, we have demonstrated that the p55 antigen elicits a cell-mediated immune response in animals previously exposed to P. carinii. Under conditions of natural exposure, the 5' portion of the molecule, p55(1-200), appears immunologically silent, failing to elicit lymphocyte proliferation or cytokine secretion. Following active immunization, the 5' portion is capable of stimulating lymphocyte proliferation. The 3' portion, p55(268-414), has at least one immunodominant region which contains a 7-amino-acid repeat motif. The cells responding to p55 include a CD4+ T cell which secretes a Th1 cytokine pattern. A detailed understanding of the host-parasite interaction will facilitate the development of immunoprophylaxis and immunotherapy for P. carinii infection.

Fractionation of Pneumocystis carinii developmental stages by counterflow centrifugal elutriation and sequential filtrations.

Immunomagnetic sorting, sequential filtrations, and counterflow centrifugal elutriation were compared for their ability to obtain enriched populations of Pneumocystis carinii developmental stages from infected rat-lung homogenates. Elutriation combined with sequential filtrations resulted in highly (> 95%) enriched populations of P. carinii cysts and trophozoites with excellent viability. This approach offers advantages over previously described methods of obtaining enriched P. carinii cell populations and should have important applications to research on this organism.

Geographic variation in the humoral response to Pneumocystis carinii.

The reported incidence of Pneumocystis carinii pneumonia varies in different areas of the world, but little is known about geographic variation in the antibody response to specific antigens. The frequency of anti-P. carinii antibodies in the serum of normal and human immunodeficiency virus (HIV)-infected individuals from five different regions was compared. Serum specimens from 948 subjects were assayed for IgG antibodies to human-derived P. carinii by Western blot. The overall prevalence of anti-P. carinii antibodies was 72.9%. Among HIV-seronegative individuals, the rate of seropositivity was similar in all regions (70.5%-82.4%). Antibodies to the 30- to 40-kDa antigen were most commonly detected. The frequency of antibodies to high-molecular-mass antigens (95, 120, and > 120 kDa) demonstrated significant geographic variation. This study demonstrates that antibody responses to P. carinii are common in all areas studied but vary in frequency and pattern.

Fatal myocardial infarction following therapy with prothrombin complex concentrates in a young man with hemophilia A.

A fatal myocardial infarction in a 22-year-old man with hemophilia A and a factor VIII inhibitor is described. The catastrophic event occurred while the patient was receiving high doses of unactivated prothrombin complex concentrates. Autopsy examination revealed myocardial hemorrhage with no evidence of coronary artery disease or thrombosis. There also was postmortem evidence of previous myocardial infarctions. This is the fourth documented case of myocardial infarction occurring in a young hemophiliac patient using unactivated prothrombin complex concentrates. It is concluded that utilization of prothrombin complex concentrates in hemophiliac patients must be limited and closely monitored. Therapeutic guidelines are recommended.

The red blood cell as a biopsy tool.

During the past several years, there has been much emphasis on understanding the relationship between abnormalities in RBC metabolism and hereditary haemolytic anaemias. More recently, attention has focused on utilizing erythrocyte biochemical abnormalities in the diagnosis of systemic disorders not necessarily associated with altered RBC function. In this chapter, we have enumerated several non-erythrocytic clinical conditions clearly associated with altered RBC metabolic products or enzyme activity. Specifically, we have described the use of erythrocytes (1) to diagnose inborn errors of metabolism, (2) to assess many nutritional disorders, (3) to monitor hyperglycaemia in patients with diabetes mellitus, and (4) to discriminate between essential and non-essential hypertension. As Eric Ponder said many years ago (Ponder, 1948), 'I have been told that I tend to speak of the red cell as if it were a microcosm, as if an understanding of its nature and properties would include an understanding of nearly everything else in the cellular world. To some extent this is true.' When the list of conditions for which the red cell can be used as a biopsy tool is reassessed several years from now, most assuredly many new clinical conditions will be added to the diseases we have discussed. For example, our knowledge of normal membrane structure and function currently is in its infancy, yet it already appears that analysis of RBC membranes may be useful in detecting certain neurological disorders such as muscular dystrophy (Roses, Herbstreith and Appel, 1975). Needless to say, to the delight of Dr Eric Ponder, the utility of the erythrocyte as an investigative tool appears to be without limit.

Erythrocyte enzyme disorders in children.

Erythrocyte metabolic abnormalities should be considered as a possible cause of hemolysis when there is no evidence of an immune-mediated hemolytic anemia, no consumptive red blood cell disorder, no morophologic or laboratory data to suggest a problem of the red cell membrane, and no evidence of a quantitative or qualitative defect in hemoglobin synthesis. Glucose-6-phosphate dehydrogenase deficiency is clearly the most common enzyme deficiency causing clinical problems.

Erythrocyte disorders leading to potassium loss and cellular dehydration.

RBC K loss and cellular dehydration are associated with a variety of normal and abnormal erythrocyte conditions. In some cases (normal RBC aging, pyruvate-kinase-deficient RBCs and irreversibly sickled cells) cation and water changes are related to adenosine triphosphate (ATP) depletion and to increased RBC calcium content. In other disorders, such as hereditary xerocytosis, cation depletion and cellular hydration are not related to altered energy or calcium metabolism. Rather, this condition is thought to be due to a structural membrane defect which is manifested by imbalanced cation leaks (K less greater than Na gain) for which the active cation transport is unable to compensate. None of the disorders described here are associated with known structural membrane alterations. The fact that K loss and cellular dehydration are common to several RBC disorders suggests that this phenomenon may have a direct role in membrane injury. This hypothesis is supported by two separate observations: 1)Formation of irreversible sickled cells in vitro is prevented if K and water loss are inhibited, and these effects are independent of ATP depletion and calcium accumulation; 2) the mean critical hemolytic volume is markedly reduced in K- and water-depleted normal RBCs. RBC dehydration without intracellular cation depletion, however, is not associated with changes in mean critical hemolytic volume. These data thus indicate that K loss may have a direct role in RBC membrane injury. The mechanism by which this occurs and the associated alterations in membrane structure, however, remain to be identified.