Dendritic cells in the induction of protective and nonprotective anticryptococcal cell-mediated immune responses.

Dendritic cells (DC) can be divided into three subsets, Langerhans cells, myeloid DC (MDC), and lymphoid DC (LDC), based upon phenotypic and functional differences. We hypothesized that different DC subsets are associated with the development of protective vs nonprotective cell-mediated immune (CMI) responses against the fungal pathogen, Cryptococcus neoformans. To test this, mice were immunized with protective and/or nonprotective immunogens, and DC subsets in draining lymph nodes were assessed by flow cytometry. The protective immunogen (cryptococcal culture filtrate Ag-CFA), in contrast to the nonprotective immunogen (heat-killed cryptococci-CFA), the nonprotective immunogen mixed with the protective immunogen (cryptococcal culture filtrate Ag + heat-killed cryptococci-CFA), or controls, stimulated significant increases in total leukocytes, Langerhans cells, MDC, LDC, and activated CD4+ T cells in draining lymph nodes. The protective immune response resulted in significantly increased levels of anticryptococcal delayed-type hypersensitivity reactivity and activated CD4+ T cells at the delayed-type hypersensitivity reaction site. Draining lymph node LDC:MDC ratios induced by the protective immunogen were significantly lower than the ratios induced by either immunization in which the nonprotective immunogen was present. In contrast, mice given the nonprotective immunogen had LDC:MDC ratios similar to those of naive mice. Our data indicate that lymph node Langerhans cells and MDC are APC needed for induction of the protective response. The predominance of LDC in mice undergoing nonprotective responses suggests that lymph node LDC, like splenic LDC, are negative regulators of CMI responses. In addition to showing DC subsets associated with functional differences, our data suggest that the LDC:MDC balance, which can be modulated by the Ag, determines whether protective or nonprotective anticryptococcal CMI responses develop.

Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4-(succinimidyl)benzophenone].

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzyme-reagent complex (K(R)) of 99 +/- 2 microM and k(max) of 0.082 +/- 0.005 min(-1). Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (Delta(5)-androstene-3,17-dione and 17beta-estradiol-3, 17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2.

[Acute meningococcemia in a 4-month-old infant]. / Akutna meningokokcemija cetvoromesecnog odojceta.

INTRODUCTION: What causes meningococcial diseases (MD) is a Gramm-negative diplococcus Neisseria meningitidis (meningococcus). Most frequently it manifests itself in the form of meningitis and meningococcemia. The mortality rate of those suffering from MD has not significantly changed for three decades and ranges from 7% to 19%, and for meningococcemia from 18% to 53%. According to the data presented by domestic authors, of the total of the diseased with bacterial meningitis 75% are children with mortality rate from 6% to 15%. Severe forms of meningococcemia sometimes have extremely rapid development and lethal outcome within a few hours. Key elements for establishing the diagnosis of meningococcemia are presence of hemorrhagic rash with high fever, loss of interest in the environment, loss of consciousness and paleness. CASE REPORT: The boy was admitted to hospital as an emergency case on 29 December 1988 at 11:45. The disease manifested abruptly the day before. He burst into tears easily, would take very little food and in the evening his temperature rose T degree 40.8 degrees C. Before midnight, in the village he was given injections of: lincomycin 300 mg and lasdol 250 i.m. During admittance the infant was agitated, kept moaning. His skin was pail gray with dot-like and spot-like hematoma which were more numerous and intense on the left ear, lower part of the body, scrotum and legs. The infant breathed heavily and fast (FR: 100/min) Cardiologist's finding showed: tahicardia over 200/min, buffled tones, gallop rhythm, pulsating neck veins and edema point to acute heart failure. Large fontanelles remained swollen even after lumbar puncture (LP) and extraction of 15 ml of clear cerebro-spinal fluid. Soon after admittance the boy stopped moaning but didn't cry when pricked and slipped deeper into coma. During the third hour of treatment generalized convulsions began which lasted approximately 10 minutes and stopped after i.v. administered diazepam. The boy remained in coma the second day in lethargy and with swollen fontanelles on the third day, so the first subdural puncture was then performed bilaterally. On that occasion only from the right side 8 ml of reddish liquid was obtained. Right after he was admitted we began permanent transfusion, which lasted 17 days. On the first day he received fresh blood transfusion. He was administered benzinpenicilline, chloramphenicol-succinate, lanatoside, human albumins, dexamethasone.... Blood oxygenation was carried out in the first few days of illness during the exhibited cardio-respiratory failure. DISCUSSION: Among risk factors, which contribute to occurrence of meningococcemia, is also artificial infant food. The reported boy was incorrectly fed with overdiluted cow milk. That and apparent hemostasis brake-down only worsened anemia and increased susceptibility to infections. Although LP was performed when the boy was admitted and the nutritious foundation was soaked with cerebro-spinal fluid, no bacteria were isolated or their presence confirmed in cerebro-spinal fluid colored after Gramm's method, because the child was given linkomycin the previous night. CONCLUSION: On the basis of clinical findings, hemorrhagic rush, convulsions, coma and acute heart failure as well as the laboratory findings it was concluded that it was a case of severe meningococcemia, meningitis and subdural effusion. Listed therapy and six subdural punctures led to full recovery of the patient. Further examination by a pediatrician and a psychologist eliminated the possibility of mental deficiency. The boy is now a good fifth grade elementary school pupil.

Enhancement of heparin cofactor II anticoagulant activity.

Heparin cofactor II (HCII) is a serpin whose thrombin inhibition activity is accelerated by glycosaminoglycans. We describe the novel properties of a carboxyl-terminal histidine-tagged recombinant HCII (rHCII-CHis(6)). Thrombin inhibition by rHCII-CHis(6) was increased >2-fold at approximately 5 microgram/ml heparin compared with wild-type recombinant HCII (wt-rHCII) at 50-100 microgram/ml heparin. Enhanced activity of rHCII-CHis(6) was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Delta1-52, Delta1-68, and Delta1-75) in wt-rHCII and rHCII-CHis(6). Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis(6) deletion mutants were less active compared with full-length rHCII-CHis(6). With glycosaminoglycans, Delta1-68 and Delta1-75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis(6), rHCII-CAla(6), and rHCII-CLys(6) to wt-rHCII. Only rHCII-CHis(6) had increased activity with heparin, whereas all three mutants have increased heparin binding. We generated a carboxyl-terminal histidine-tagged recombinant antithrombin III to study the tag on another serpin. Interestingly, this mutant antithrombin III had reduced heparin cofactor activity compared with wild-type protein. In a plasma-based assay, the glycosaminoglycan-dependent inhibition of thrombin by rHCII-CHis(6) was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis(6), may offer novel reagents for clinical application.

Photoaffinity labeling of rat liver glutathione S-transferase, 4-4, by glutathionyl S-[4-(succinimidyl)-benzophenone].

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and xenobiotic substrate, was synthesized and shown to act as a photoaffinity label of rat liver glutathione S-transferase, 4-4. A time-dependent photoinactivation occurs upon irradiation at long wavelength UV light of the complex of enzyme and GS-Succ-BP. The rate of inactivation exhibits nonlinear dependence on [GS-Succ-BP], characterized by an apparent KI of 115 microM and kmax of 0.469 min-1. Effective protection against photoinactivation by 150 microM GS-Succ-BP is provided by dinitrophenol, nitrobenzene, ethacrynic acid, and S-hexylglutathione, analogues of xenobiotic substrates and product. These results suggest that GS-Succ-BP reacts with the enzyme within the active site, probably in the xenobiotic substrate-binding site. Upon complete inactivation, reagent incorporation of about 1 mol/mol of enzyme dimer is measured by radioactivity and MALDI-TOF mass spectrometry. Isolation of modified peptides followed by gas-phase sequencing and mass spectrometry indicates that Met-112 is the only reaction target of GS-Succ-BP. Although only one subunit of the enzyme dimer is modified, catalytic activity of both subunits is lost. Molecular modeling suggests that the benzophenone moiety of the compound binds in the cleft between the two enzyme subunits and modification of Met-112 on one subunit excludes reaction of the corresponding methionine on the other subunit. It is proposed that the new compound, glutathionyl S-[4-(succinimidyl)benzophenone], may have general applicability as a photoaffinity label of other enzymes with glutathione binding sites.

Physical aspects of the self. A review of some aspects of body image development in childhood.

The concept of body image in psychiatric usage, although vague as a concept, is nonetheless valuable in understanding the effects on development of a child's physical defect or physical illness, or of illness in the mother. The concepts of mirroring and the development of a true self have been considered as they relate to the body. Finally, a case of anorexia nervosa in a young adolescent, wherein mirroring of the physical self by the mother was aberrant in part because of an illness in infancy, has been presented. The development of a cohesive physical self in childhood is an important area for future theoretical and clinical work which may advance our understanding of the treatment process in some children and adults.