Randomized, Placebo Controlled Trial of NPT088, A Phage-Derived, Amyloid-Targeted Treatment for Alzheimer's Disease.

The engineered fusion protein NPT088 targets amyloid in vitro and in animal models of Alzheimer's disease. Previous studies showed that NPT088 treatment reduced ß-amyloid plaque and tau aggregate loads in mouse disease models. Here, we present the results from an initial clinical study of NPT088 in patients with mild to moderate Alzheimer's disease. Patients were treated with 4 dose levels of NPT088 for 6 months to evaluate its safety and tolerability. Exploratory measurements included measurement of change in ß-amyloid plaque and tau burden utilizing Positron Emission Tomography imaging as well as measures of Alzheimer's disease symptoms. At endpoint NPT088 was generally safe and well-tolerated with the most prominent finding being infusion reactions in a minority of patients. No effect of NPT088 on brain plaques, tau aggregates or Alzheimer's disease symptoms was observed.

Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.

Pulse oximetry screening for critical congenital heart disease in the neonatal intensive care unit.

OBJECTIVE: Pulse oximetry screening (POS) is an effective tool to detect critical congenital heart disease (CCHD) in asymptomatic term infants, but its value in the neonatal intensive care unit (NICU) requires further clarification. STUDY DESIGN: A retrospective review of 1005 babies without previously diagnosed CCHD admitted to a level III NICU was performed to assess the risk for missed CCHD and performance of POS. RESULT: Of the 1005 NICU patients, 812 had documented POS and none failed POS. In 812 patients, 547 had delayed POS because of the use of supplemental oxygen. In 259/812 patients, POS was delayed until the baby was >2 weeks old. CCHD was excluded by echocardiography, irrespective of POS, in 287/1005 patients. CONCLUSION: POS can be performed in the NICU with minimal adverse effects. However, in many NICU patients CCHD is confirmed or excluded before POS, and POS will frequently be performed after CCHD would have been expected to become symptomatic.

Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli.

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. Malonyl transacylase has been overexpressed 155-fold compared to a wild-type strain. Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of cis-vaccenate are incorporated into the membrane phospholipids.

Myringotomy: a prerequisite for the development of myringosclerosis?

Streptococcus pneumoniae was inoculated into the left middle-ear cavity in two groups of rats, resulting in purulent otitis media. After 3 days, one group of infected animals and a third group of noninfected animals were subjected to left-sided myringotomy. The tympanic membranes were examined both otomicroscopically and histologically 1 and 3 months later. On otomicroscopic examination the noninfected myringotomized animals had developed extensive myringosclerotic lesions, whereas only minimal sclerotic deposits were noted in the myringotomized animals with acute otitis media (AOM). On histologic examination both the noninfected myringotomized animals and the myringotomized animals with AOM were similar in the frequency and extension of sclerotic lesions in the tympanic membrane. The nonmyringotomized rats with AOM were free of sclerotic lesions, except for minor changes found in one animal.

Myringosclerosis caused by increased oxygen concentration in traumatized tympanic membranes. Experimental study.

The purpose of this study was to elucidate possible relationships between the oxygen concentration of the middle ear cavity and the development of myringosclerosis. Three groups of rats with myringotomized tympanic membranes were exposed to different oxygen concentrations of 10%, 15%, and 40%, respectively, for 1 week. A fourth group was kept in ambient air. Two other groups of rats with myringotomized and intubated tympanic membranes were exposed to oxygen concentrations of 10% and 40%, respectively, for the same period of time. Otomicroscopically, all hyperoxic animals had more numerous myringosclerotic lesions compared with the ambient air group, and also displayed a pronounced hyperplasia of the keratinizing epithelium around the perforation border. By contrast, the hypoxic animals showed less pronounced myringosclerotic lesions or even completely lacked them. It is inferred that an increased oxygen concentration in the middle ear cavity will increase the likelihood of myringosclerotic deposits. The mechanism involved could be related to the formation of oxygen radicals.

Healing of tympanic membrane after myringotomy during Streptococcus pneumoniae otitis media. An otomicroscopic and histologic study in the rat.

The purpose of our study was to elucidate the course of healing of the tympanic membrane (TM) when myringotomy was performed during acute otitis media. The early and long-lasting structural changes of the TM were studied in an animal model. Rats were inoculated with Streptococcus pneumoniae (PnC) type 3 in the bulla. When the infection was manifest, myringotomy was performed. On days 4 and 12, and 3 and 6 months after myringotomy, the TM status was checked by otomicroscopy and TMs were prepared for light and electron microscopy. Comparison was made with PnC-infected TMs that were not perforated, as well as myringotomized noninfected TMs. The infection resolved more slowly in myringotomized ears compared to PnC-infected ears that were left untouched. After 6 months, the pars tensa of the myringotomized infected ears was thickened and showed a disorganized collagen structure, compared with myringotomized noninfected ears, in which TMs were normalized. The PnC-infected TMs without myringotomy were completely normalized after 2 months. We conclude that a combination of bacterial infection and myringotomy causes long-lasting changes in TM structure. This impaired structure of the connective tissue could be of importance in chronic middle ear disease as a presumptive site for retraction and perforation of the TM.

The tympanic membrane and middle ear mucosa during non-typeable Haemophilus influenzae and Haemophilus influenzae type b acute otitis media: a study in the rat.

Non-typeable Haemophilus influenzae (NTHi) and encapsulated Haemophilus influenzae type b (Hib) were inoculated into the middle ears of Sprague-Dawley rats. Tympanic membrane (TM) status was assessed otomicroscopically and specimens from various middle ear areas were prepared for light microscopy at various times during the acute phase and up to 6 months after inoculation. Irrespective of bacteria strain, acute otitis media (AOM) was present in all ears 4 days after inoculation. The Hib-infected ears showed initially a severe course of AOM, but all were otomicroscopically resolved by day 12, at which time a few NTHi-inoculated ears still exhibited middle ear effusion. The TMs infected with Hib had normalized without scar formation, whereas NTHi induced a persistent thickening of the TMs in half of all cases. The middle ear mucosa of NTHi-infected ears initially showed vigorous activity among the goblet cells, but the mucosa normalized after the acute phase. Hib, by contrast, induced prominent changes in the middle ear mucosa. Initially, no goblet cell granules or ciliated cells could be observed in the mucosa. Later on, the epithelium contained large, active goblet cells. Glands appeared beneath the mucosa which persisted as streaks of epithelial cells throughout the study period. The findings show that NTHi and Hib both induce AOM but with differing clinical courses, and affect different targets in the middle ear.

Early structural changes in the rat tympanic membrane during pneumococcal otitis media.

The early inflammatory reaction in the rat tympanic membrane was studied during the first 36h following inoculation of Streptococcus pneumoniae type 3 in the middle ear cavity. Otomicroscopic examination showed only minor signs of inflammation in the early stages although changes at the light microscopic level were pronounced. This reaction differed significantly between the pars flaccida and pars tensa of the tympanic membrane. Three hours after inoculation, edema and infiltration with polymorphonuclear leukocytes and macrophages were found in the pars flaccida whereas in the pars tensa no polymorphonuclear leukocytes were noted until after 12h. This reaction was most prominent after 36h. In the pars flaccida, mitoses occurred frequently among the cells of the simple squamous epithelium, which changed into a double-layered cuboidal epithelium. These findings demonstrate that an inflammatory reaction starts earlier in the pars flaccida than in the pars tensa of the tympanic membrane.

Structural changes in the rat tympanic membrane following repeated pressure loads.

Healthy adult laboratory rats were exposed to alternating negative pressure and atmospheric pressure to replicate the clinical situation found in patients with chronic sniffing habits and chronic middle ear disease. The rats were placed in a box in which the pressure changed at intervals of 30 s between atmospheric pressure and a negative pressure of -3 kPa. This was repeated continuously for periods of 3 and 7 days. At completion of the experimental period, all rats had a normal otomicroscopic status. However, histological studies demonstrated that the pars flaccida was wrinkled and the loose connective tissue contained large fibroblasts with their long axes lying in a disorganized manner. The cells of the keratinizing epithelium were thicker than normal and mitoses were seen. Epithelial crypts filled with keratin were numerous along the epithelium. In the pars tensa, all layers were thicker than normal. These findings demonstrate that repeated pressure loading can create structural changes in the tympanic membrane.

The putative fabJ gene of Escherichia coli fatty acid synthesis is the fabF gene.

Siggaard-Andersen and coworkers (M. Siggaard-Andersen, M. Wissenbach, J. Chuck, I. Svendsen, J. G. Olsen, and P. von Wettstein-Knowles, Proc. Natl. Acad. Sci. USA 91:11027-11031, 1994) recently reported the DNA sequence of a gene encoding a beta-ketoacyl-acyl carrier protein synthase from Escherichia coli. These workers assigned this gene the designation fabJ and reported that the gene encoded a new beta-ketoacyl-acyl carrier protein synthase. We report that the fabJ gene is the previously reported fabF gene that encodes the known beta-ketoacyl-acyl carrier protein synthase II.

Structural changes in the middle ear tissues of the rat after fractionated irradiation.

Chronic suppurative otitis media often ensues in patients treated with irradiation against a head and neck tumor. In an experimental study, rats were exposed to irradiation to evaluate the sensitivity of the middle ear to an accumulated irradiation dose of 20-45 Gy. Observed otomicroscopically, all animals appeared to have normal tympanic membranes and no fluid developed in the middle ear space. Ten days after the irradiation, minor structural changes had occurred in the pars flaccida. The keratinizing epithelium had thickened and mitoses were seen histologically. The lamina propria was edematous and contained polymorphonuclear cells and macrophages. The middle ear mucosa from all other tissue sites appeared normal. Six months after irradiation only minor changes in the pars flaccida were evident: the lamina propria was thin and inelastic and macrophages were present in the stroma. It is inferred from this study that the middle ear of the rat is relatively resistant to irradiation.

Regulation of fatty acid biosynthesis in Escherichia coli.

Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.

Work-based antipoverty programs for parents can enhance the school performance and social behavior of children.

We assess the impact of the New Hope Project, an antipoverty program tested in a random assignment experimental design, on family functioning and developmental outcomes for preschool- and school-aged children (N = 913). New Hope offered wage supplements sufficient to raise family income above the poverty threshold and subsidies for child care and health insurance to adults who worked full-time. New Hope had strong positive effects on boys' academic achievement, classroom behavior skills, positive social behavior, and problem behaviors, as reported by teachers, and on boys' own expectations for advanced education and occupational aspirations. There were not corresponding program effects for girls. The child outcomes may have resulted from a combination of the following: Children in New Hope families spent more time in formal child care programs and other structured activities away from home than did children in control families. New Hope parents were employed more, had more material resources, reported more social support, and expressed less stress and more optimism about achieving their goals than did parents in the control sample. The results suggest that an anti-poverty program that provides support for combining work and family responsibilities can have beneficial effects on the development of school-age children.

Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity.

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, gammac and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

Unexpected effects of FERM domain mutations on catalytic activity of Jak3: structural implication for Janus kinases.

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.