Selective CD28 blockade attenuates acute and chronic rejection of murine cardiac allografts in a CTLA-4-dependent manner.

Selective blockade of CD28 is a promising therapy to inhibit pathogenic alloimmunity. However, evaluation of this approach in transplantation has been very limited. Using a novel nonactivating single-chain Fv-based reagent (α28scFv), we have investigated the role of CD28 and cytotoxic T lymphocyte antigen 4 (CTLA-4) in a murine cardiac transplant model. Blockade of CD28 for 2 weeks after engraftment promoted allograft survival, and significantly attenuated chronic rejection when combined with transient CD154-blockade or calcineurin inhibition. Graft acceptance was associated with decreased alloantibody production, increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Blockade of CTLA-4 during α28scFv-based treatments led to prompt rejection in all animals and inhibited expression of forkhead box P3 (Foxp3), programmed death (PD)-1 and 2,3-indoleamine dioxygenase (IDO) in the graft. These results show that CD28 signaling during the first weeks after transplant is a pivotal mediator of pathogenic alloimmunity, and that selective CD28 blockade prolongs graft acceptance by at least two immunomodulatory mechanisms. Selective CD28 inhibition while sparing CTLA-4 is thus a promising approach to inhibit pathogenic alloimmunity.

Improved Norway spruce somatic embryo development through the use of abscisic acid combined with activated carbon.

The combination of abscisic acid (ABA) and activated carbon increased Norway spruce (Picea abies L., Karst.) cotyledonary somatic embryo yields, increased the number of genotypes forming cotyledonary embryos, caused embryos to form that exhibited improved maturation characteristics, and reduced embryo production costs. Somatic embryos increased in size, showed larger apical regions, became more zygotic-like in shape, and showed higher percentages of epicotyl development upon germination. Analyses of medium for free ABA in the presence of activated charcoal showed a rapid decrease within a few hours followed by a gradual decline over the next few days with little change from 2 to 6 weeks. Gelling agents strongly affected ABA adsorption, with agar decreasing the adsorption of ABA compared to gellan gum (Gelrite, Phytagel). Over 4,000 somatic seedlings from 20 clones were produced and established in a greenhouse using the methods discussed, and approximately 1,250 seedlings representing seven clones were established in a field setting.

Differentiating the use of gaze in bilingual-bimodal language acquisition: a comparison of two sets of twins with deaf parents.

Signed languages make unique demands on gaze during communication. Bilingual children acquiring both a spoken and a signed language must learn to differentiate gaze use for their two languages. Gaze during utterances was examined for a set of bilingual-bimodal twins acquiring spoken English and American Sign Language (ASL) and a set of monolingual twins acquiring ASL when the twins were aged 2;0, 3;0 and 4;0. The bilingual-bimodal twins differentiated their languages by age 3;0. Like the monolingual ASL twins, the bilingual-bimodal twins established mutual gaze at the beginning of their ASL utterances and either maintained gaze to the end or alternated gaze to include a terminal look. In contrast, like children acquiring spoken English monolingually, the bilingual-bimodal twins established mutual gaze infrequently for their spoken English utterances. When they did establish mutual gaze, it occurred later in their spoken utterances and they tended to look away before the end.

The special role of rimes in the description, use, and acquisition of English orthography.

The links between spellings and sounds in a large set of English words with consonant-vowel-consonant phonological structure were examined. orthographic rimes, or units consisting of a vowel grapheme and a final consonant grapheme, had more stable pronunciations than either individual vowels or initial consonant-plus-vowel units. In 2 large-scale studies of word pronunciation, the consistency of pronunciation of the orthographic rime accounted for variance in latencies and errors beyond that contributed by the consistency of pronunciation of the individual graphemes and by other factors. In 3 experiments, as well, children and adults made more errors on words with less consistently pronounced orthographic rimes than on words with more consistently pronounced orthographic rimes. Relations between spellings and sounds in the simple monomorphemic words of English are more predictable when the level of onsets and rimes is taken into account than when only graphemes and phonemes are considered.

What happened to the "n" of sink? Children's spellings of final consonant clusters.

Children's spellings provide a window on their representations of spoken words. These representations may not always match those assumed by the conventional orthography. We examined one specific case in which this may be true, that of CVCC (consonant-vowel-consonant-consonant) syllables. In three experiments, first graders often produced spellings such as "sach" for the nonword [symbol: see text] and "pit" for the nonword /pIlt/. We suggest that many children consider these syllables to contain three phonemes instead of four phonemes. Rather than treating the postvocalic nasal or liquid as a separate phoneme, children consider it an attribute of the vowel. This suggestion was supported by the results of a phoneme counting test in which children often used three tokens for syllables such as [symbol: see text] and /pIlt/. The results suggest that, in addition to difficulties in gaining access to phonemes, nonstandard phonemic representations may be a stumbling block in the acquisition of alphabetic literacy.

Human exposure to polychlorinated biphenyls at toxic waste sites: investigations in the United States.

Beginning in 1982, environmental and population data were evaluated from waste sites contaminated with polychlorinated biphenyls (PCBs). Pilot exposure assessment studies were conducted at 12 sites where risks of human exposure were thought to be greatest. Serum PCB levels in persons at highest risk of nonoccupationally related exposures (because of their self-reported frequencies and types of activities in contaminated areas) at 10 sites were within background ranges, even though environmental contamination levels as high as 2.5 parts per billion (ppb) in monitoring well water samples and 330,000 ppb in soil samples were measured. At the 2 remaining sites, elevated serum levels were found in these high-risk persons, which require further evaluation by community surveys. These results illustrate that, despite elevated environmental contaminant levels, unless uptake of chemicals above background exposure levels can be demonstrated, adverse health effects cannot be attributed to waste site chemicals. However, health risks due to background exposure levels, as well as in populations with elevated PCB body burdens need further study.

HbXL99 alpha: a hemoglobin derivative that is cross-linked between the alpha subunits is useful as a blood substitute.

Under deoxygenated conditions, bis(3,5-dibromosalicyl) fumarate reacts with hemoglobin selectively to cross-link the alpha subunits between Lys-alpha 1 99 and Lys-alpha 2 99. We have characterized further the properties of this recently described hemoglobin and have demonstrated its utility as a blood substitute. The oxygen transport characteristics of the cross-linked derivative are very similar to those of whole blood. Under physiological conditions, the partial pressure of oxygen at half-saturation of hemoglobin is increased to 29 mm Hg (1 mm Hg = 133.3 kPa), compared to 12 mm Hg for hemoglobin A, fully compensating for the absence of 2,3-bisphosphoglycerate outside of the erythrocyte. The Hill coefficient is 2.9. The dependence of the oxygen affinity of HbXL99 alpha on CO2 is also identical to that of hemoglobin A. The cross-link between the alpha subunits blocks dissociation of oxyhemoglobin into alpha beta dimers and thereby prevents renal excretion of the modified hemoglobin. In the rat, the half-life of HbXL99 alpha in plasma, at a 15% volume exchange, is increased to 3.3 hr, compared to 90 min for hemoglobin A. Cross-linking HbXL99 alpha intermolecularly with bis(sulfosuccinimidyl) suberate to form predominantly a mixture of dimers and trimers further increased the half-life of the hemoglobin within the circulation by about 2-fold. The rate of autooxidation of the transfused hemoglobin was found to be markedly reduced because of the presence of an endogenous reducing system in plasma.

Evaluation of potential health effects associated with serum polychlorinated biphenyl levels.

In late 1983, we conducted a cross-sectional epidemiologic study to evaluate persons at risk of exposure to three chemical waste sites by comparing clinical disease end points and clinical chemistry parameters with serum polychlorinated biphenyls (PCB) levels. A total of 106 individuals participated in the study. The only statistically significant finding in regard to self-reported, physician-diagnosed health problems was a dose-response relationship between serum PCB levels and the occurrence of high blood pressure; however, this association failed to achieve statistical significance (p = 0.08) when we controlled for possible confounding effects of both age and smoking. Serum triglyceride and cholesterol levels were also higher in the group with elevated serum PCBs; additionally, there were isolated statistically significant correlations of serum aspartate aminotransferase (SGOT) with serum lipid fraction-adjusted PCB level (r = -0.21) and serum albumin (r = -0.24) and total bilirubin (r = 0.30) with serum PCB level. Although the ranges of serum levels reported herein from exposures to PCBs in the general environment are lower than those that have been associated with acute symptoms or illness in other studies, whether these levels are associated with long-term health risks is not known. Associations of such chronic, low-dose exposures with observable health effects as suggested by this study must be evaluated further before any final conclusions can be drawn.

The effect of 2,3-diphosphoglycerate on the solubility of deoxyhemoglobin S.

Although highly charged polyanions, such as inositol hexaphosphate, have been clearly shown to decrease the solubility of deoxyhemoglobin S, the effect of 2,3-diphosphoglycerate (DPG), the endogenous allosteric effector within the red cell, has been more controversial. In this work we have compared the effect of DPG on the solubility of native deoxyhemoglobin S and a derivative in which the DPG binding site is blocked by cross-linking the two beta 82 lysine residues. At pH 6.6 and 30 degrees C the solubility of deoxyhemoglobin S was found to be decreased by 15% (i.e., from 18.8 to 16.0 g/dl) in the presence of saturating concentrations of DPG. Under the same conditions DPG had no effect on the solubility of the cross-linked derivative. This result establishes unequivocally that the binding of DPG within the beta cleft directly facilitates the polymerization of deoxyhemoglobin S. Under physiological conditions, the solubility of deoxyhemoglobin S was found to be decreased by 6% in the presence of an equimolar concentration of DPG. A solubility decrease of this magnitude is sufficient to enhance the tendency of SS cells to sickle and may exacerbate the clinical symptoms of sickle cell disease.

Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2).

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.

A pilot study of serum polychlorinated biphenyl levels in persons at high risk of exposure in residential and occupational environments.

A pilot study was conducted to determine whether persons at high risk of exposure to three waste sites in the area of Bloomington, Indiana, have abnormally elevated serum polychlorinated biphenyl (PCB) levels. In addition, we attempted to determine which environmental pathways might have contributed most to these exposures. First, a screening questionnaire survey of 995 individuals was conducted; on the basis of these data, 114 of the persons who had the greatest potential for exposure were selected for inclusion in this pilot exposure assessment study. People near these waste sites have higher average serum PCB levels, and a greater percentage have abnormally elevated serum PCB levels, compared with previously characterized populations in the United States. However, we could not distinguish specific pathways of exposure and uptake, with the exception of persons with occupational exposures and, possibly, among persons who reportedly salvaged metal from discarded electrical equipment. Exposures in this community require further evaluation.

Nineteen cases of plague in Arizona. A spectrum including ecthyma gangrenosum due to plague and plague in pregnancy.

We review the cases of 19 successfully treated plague patients, with emphasis on the clinical and epidemiologic features of the disease. Proper staining and culturing of bubo aspirates; prompt institution of streptomycin, chloramphenicol or tetracycline therapy in presumptive cases, and supportive care are the crucial factors in the treatment of plague. This disease should be considered in patients in a toxic condition who have lymphadenitis, pneumonia or septic shock and who have been in endemic areas within the past ten days.