A Dynamic Model for the Evolution of Protein Structure.

Domains are folded structures and evolutionary building blocks of protein molecules. Their three-dimensional atomic conformations, which define biological functions, can be coarse-grained into levels of a hierarchy. Here we build global dynamical models for the evolution of domains at fold and fold superfamily (FSF) levels. We fit the models with data from phylogenomic trees of domain structures and evaluate the distributions of the resulting parameters and their implications. The trees were inferred from a census of domain structures in hundreds of genomes from all three superkingdoms of life. The models used birth-death differential equations with the global abundances of structures as state variables, with one set of equations for folds and another for FSFs. Only the transitions present in the tree are assumed possible. Each fold or FSF diversifies in variants, eventually producing a new fold or FSF. The parameters specify rates of generation of variants and of new folds or FSFs. The equations were solved for the parameters by simplifying the trees to a comb-like topology, treating branches as emerging directly from a trunk. We found that the rate constants for folds and FSFs evolved similarly. These parameters showed a sharp transient change at about 1.5 Gyrs ago. This time coincides with a period in which domains massively combined in proteins and their arrangements distributed in novel lineages during the rise of organismal diversification. Our simulations suggest that exploration of protein structure space occurs through coarse-grained discoveries that undergo fine-grained elaboration.

Delta chemokine (fractalkine)--a novel mediator of pulmonary arterial hypertension in children undergoing cardiac surgery.

BACKGROUND: Fractalkine (FKN), a unique chemokine associated with pulmonary hypertension, may be involved in the acute stress response that regulates inflammation after cardiopulmonary bypass (CPB) surgery. We characterized FKN levels and correlated them with clinical parameters in children undergoing cardiac surgery involving CPB. METHODS: Twenty-seven consecutive patients, aged 30 days to 11.5 years, who underwent surgery for correction of congenital heart defects, were prospectively studied. Serial blood samples were collected preoperatively, upon termination of CPB, and at six points postoperatively. Plasma was recovered immediately, aliquoted, and frozen at -70° C until assayed. Clinical and laboratory data were collected. RESULTS: Baseline FKN levels were skewed between patients. Patients with low FKN levels showed significantly higher levels of oxygen saturation in room air compared to patients with high FKN levels (p<0.05). Moreover, there was a positive correlation between preoperative pulmonary arterial hypertension and FKN levels (p<0.05). Surprisingly, FKN elevation from preoperative to postoperative levels displayed no discernible pattern. CONCLUSIONS: FKN levels significantly correlate with preoperative hypoxemia and PAH, suggesting that FKN may be up-regulated during hypoxemia. CPB is not associated with acute changes in circulating FKN levels. The role of FKN in the postoperative course should be further investigated.

Anti-inflammatory effects of an inflammatory chemokine: CCL2 inhibits lymphocyte homing by modulation of CCL21-triggered integrin-mediated adhesions.

Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1- and LFA-1-mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.

Does high serum progesterone level on the day of human chorionic gonadotropin administration affect pregnancy rate after intracytoplasmic sperm injection and embryo transfer?

OBJECTIVE: The present study was conducted to evaluate the effect of serum progesterone (P) levels on the day of human chorionic gonadotropin (hCG) administration on embryo quality and pregnancy rate in intracytoplasmic sperm injection (ICSI) cycles. DESIGN AND SETTING: This was a retrospective analysis conducted in the in vitro fertilization (IVF) unit of a tertiary hospital. PATIENTS: Two hundred and one patients who underwent a total of 280 IVF treatment cycles allocated to ICSI during routine IVF/embryo transfer treatment. Results. In cycles with elevated serum P, higher estradiol levels were noted (1915 pg/ml vs. 1256 pg/ml; p<0.05), more oocytes were retrieved and manipulated, and more embryos were available for transfer. Embryo grading was comparable between the two groups. The average age was lower in the group with elevated P; but the pregnancy rate was significantly lower (16.4% vs. 27.6%, p = 0.03). CONCLUSIONS: Our data demonstrate no deleterious effect of elevated P on embryo quality. However, high serum P adversely affects implantation and pregnancy rates.

Epidemiologic, socioeconomic, and clinical factors associated with severity of respiratory syncytial virus infection in previously healthy infants.

We prospectively quantified disease severity associated with epidemiologic and socioeconomic parameters as well as the clinical factors in 195 previously healthy infants with confirmed respiratory syncytial virus (RSV) infection. Infants were enrolled into three subgroups according to disease severity: outpatients (82 patients), inpatients (100 patients), and intensive care unit patients (13 patients). Epidemiologic parameters such as gestational age, birth weight, chronologic age at presentation, and gender as well as socioeconomic factors such as ethnic origin, family history of asthma, exposure to cigarette smoke, number of family members, presence of pets at home, breast-feeding, and day-care attendance were not found to predict the severity of RSV illness in previously healthy infants. Our results emphasize the complexity of predicting disease severity in previously healthy infants with RSV infection and suggest that other parameters such as host genetic background might explain the clinical variability.

Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling.

CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25- or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-gamma and TNF-alpha secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-beta and IL-10. In addition, the expression of ERK, NF-kappaB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling.

Hypertonic saline/epinephrine treatment in hospitalized infants with viral bronchiolitis reduces hospitalization stay: 2 years experience.

BACKGROUND: We recently published preliminary evidence on the effectiveness of hypertonic saline in infants with viral bronchiolitis. OBJECTIVE: To further establish the efficacy of nebulized hypertonic saline in these infants. METHODS: In a continuing, second-year randomized, doubleblind controlled trial, an additional 41 infants (age 2.6 +/- 1 months) hospitalized with viral bronchiolitis were recruited during the winter of 2001-2002. The infants received inhalation of 1.5 mg epinephrine dissolved either in 4 ml normal (0.9%) saline (Group I, n=20) or 4 ml hypertonic (3%) saline (Group II, n=22). The therapy was repeated three times daily until discharge. Pooling our 2 years of experience (2000-2002), a total of 93 hospitalized infants with viral bronchiolitis were recruited; 45 were assigned to Group I and 48 to Group II. RESULTS: The clinical scores at baseline were 7.6 +/- 0.7 for Group I vs. 7.4 +/- 1.3 for Group II (P = NS). However, the clinical scores at days 1 and 2 after inhalation differed significantly between the two groups, invariably favoring Group II: 7 +/- 1 vs. 6.25 +/- 1.1 (P< 0.05), 6.45 +/- 1 vs. 5.35 +/- 1.35 (P< 0.05), respectively. Adding aerosolized 3% saline to 1.5 mg epinephrine reduced the hospitalization stay from 3.5 +/- 1.7 days in Group I to 2.6 +/- 1.4 in Group II (P< 0.05). The pooled data of both years revealed that adding 3% saline to the inhalation mixture decreased hospitalization stay from 3.6 +/- 1.6 to 2.8 +/- 1.3 days (P< 0.05). CONCLUSIONS: This second-year experience and our 2 year pooled data analysis strengthen the evidence that the combination of 3% saline/1.5 mg epinephrine benefits hospitalized infants with viral bronchiolitis.

Aerosol delivery in respiratory syncytial virus bronchiolitis: hood or face mask?

OBJECTIVES: To compare the utility of the hood versus the face mask for delivery of inhaled medications to infants hospitalized with viral bronchiolitis. STUDY DESIGN: Randomized, double-blinded, controlled trial; 49 hospitalized infants with viral bronchiolitis, age 2.75 +/- 2.2 months (mean +/- SD), were grouped to either the hood (n = 25) or the mask (n = 24). Each subject received inhalation treatments with the use of both devices. Half of the Hood Group received the active drug treatment (1.5 mg epinephrine in 4 mL saline [3%]) via hood followed immediately by placebo treatment (normal saline) via mask, whereas the other half received the opposite order. Half of the Mask Group received the active drug treatment via mask followed immediately by placebo treatment via hood, whereas the other half received the opposite order. Therapy was repeated 3 times daily until discharge. Outcome measures included clinical scores and parental preference. RESULTS: Percent improvement in clinical severity scores after inhalation was significant in both groups on days 1, 2, and 3 after admission (Hood Group: 15%, 15.4%, and 16.4%, respectively; Mask Group: 17.5%, 12.1%, and 12.7%, respectively; P < .001). No significant difference in clinical scores improvement between groups was observed. Eighty percent (39/49) of parents favored the hood over the mask; 18% (9/49) preferred the mask and 2% (1/49) were indifferent. CONCLUSIONS: In infants hospitalized with viral bronchiolitis and in whom aerosol treatment is considered, aerosol delivery by hood is as effective as by mask. However, according to parents, the tolerability of the hood is significantly better than that of a mask.

Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2.

The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not HBc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-alpha, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-kappaB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-kappaB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.

Heat shock protein 60 activates cytokine-associated negative regulator suppressor of cytokine signaling 3 in T cells: effects on signaling, chemotaxis, and inflammation.

Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1alpha (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1alpha and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation.

Heat shock protein 60 inhibits Th1-mediated hepatitis model via innate regulation of Th1/Th2 transcription factors and cytokines.

Extracellular heat shock protein 60 (HSP60) has been considered a proinflammatory danger signal. Yet, HSP60 can also down-regulate experimental immune arthritis and diabetes models by specific inhibition of Th1-like responses. We now report that HSP60 in vitro differentially modulates the expression of Th1/Th2 transcription factors in human T cells: HSP60 down-regulates T-bet, NF-kappaB, and NFATp and up-regulates GATA-3, leading to decreased secretion of TNF-alpha and IFN-gamma and enhanced secretion of IL-10. These effects depended on TLR2 signaling and could not be attributed to LPS or to other contaminants. In BALB/c mice, HSP60 in vivo inhibited the clinical, histological, and serological manifestations of Con A-induced hepatitis associated with up-regulated T cell expression of suppressor of cytokine signaling 3 and GATA-3 and down-regulated T-bet expression. These results provide a molecular explanation for the effects of HSP60 treatment on T cell inflammation via innate regulation of the inflammatory response.

Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease.

BACKGROUND: The clinical spectrum of respiratory syncytial virus (RSV) bronchiolitis in previously healthy infants is extremely variable. Thus, it is likely that factors such as genetic heterogeneity contribute to disease severity. Toll-like receptor 4 (TLR4) and CD14 are part of a receptor complex involved in the innate immune response to RSV. METHODS: The association of the TLR4 mutations (Asp299Gly and Thr399Ile) and the CD14/-159 polymorphism were analyzed in 99 infants hospitalized with severe RSV bronchiolitis (group I). Eighty-two ambulatory infants with mild RSV bronchiolitis (group II) and 90 healthy adults (group III) composed the 2 control groups. The TLR4 mutations and the CD14/-159 polymorphism were genotyped by use of reverse-transcriptase polymerase chain reaction and restriction fragment-length polymorphism analysis, respectively. RESULTS: Each of the TLR4 mutations, either alone or in cosegregation, were associated with severe RSV bronchiolitis: the Asp299Gly and Thr399Ile mutations were significantly overrepresented in group I, compared with groups II and III. No association between the CD14/-159 polymorphism and RSV bronchiolitis was found. CONCLUSIONS: These findings suggest that TLR4 mutations, but not the CD14/-159 polymorphism, are associated with an increased risk of severe RSV bronchiolitis in previously healthy infants.

Nebulized 3% hypertonic saline solution treatment in hospitalized infants with viral bronchiolitis.

OBJECTIVE: To determine the utility of inhaled hypertonic saline solution to treat infants hospitalized with viral bronchiolitis. DESIGN: Randomized, double-blind, controlled trial. Fifty-two hospitalized infants (mean +/- SD age, 2.9 +/- 2.1 months) with viral bronchiolitis received either inhalation of epinephrine, 1.5 mg, in 4 mL of 0.9% saline solution (group 1; n = 25) or inhalation of epinephrine, 1.5 mg, in 4 mL of 3% saline solution (group 2; n = 27). This therapy was repeated three times every hospitalization day until discharge. RESULTS: The percentage improvement in the clinical severity scores after inhalation therapy was not significant in group 1 on the first, second, and third days after hospital admission (3.5%, 2%, and 4%, respectively). In group 2, significant improvement was observed on these days (7.3%, 8.9%, and 10%, respectively; p < 0.001). Also, the improvement in clinical severity scores differed significantly on each of these days between the two groups. Using 3% saline solution decreased the hospitalization stay by 25%: from 4 +/- 1.9 days in group 1 to 3 +/- 1.2 days in group 2 (p < 0.05). CONCLUSIONS: We conclude that in nonasthmatic, nonseverely ill infants hospitalized with viral bronchiolitis, aerosolized 3% saline solution/1.5 mg epinephrine decreases symptoms and length of hospitalization as compared to 0.9% saline solution/1.5 mg epinephrine.

Nebulized 3% hypertonic saline solution treatment in ambulatory children with viral bronchiolitis decreases symptoms.

OBJECTIVE: To determine the utility of inhaled hypertonic saline solution to treat ambulatory infants with viral bronchiolitis. DESIGN: Randomized, double-blind, controlled trial. Sixty-five ambulatory infants (mean +/- SD age, 12.5 +/- 6 months) with viral bronchiolitis received either of the following: inhalation of 0.5 mL (5 mg) terbutaline added to 2 mL of 0.9% saline solution as a wet nebulized aerosol (control; group 1; n = 32) or 0.5 mL (5 mg) terbutaline added to 2 mL of 3% saline solution administered in the same manner as above (treatment; group 2; n = 33). This therapy was repeated three times every day for 5 days. RESULTS: The clinical severity (CS) scores at baseline on the first day of treatment were 6.4 +/- 1.8 in group 1 and 6.6 +/- 1.5 in group 2 (not significant). After the first day, the CS score was significantly lower (better) in group 2 as compared to group 1 on each of the treatment days (p < 0.005; Fig 1 ). On the first day, the percentage decrease in the CS score after inhalation therapy was significantly better for group 2 (33%) than for group 1 (13%) [p < 0.005; Fig 1 ]. On the second day, the percentage improvement was better in the hypertonic saline solution-treated patients (group 2) as compared to the 0.9% saline solution-treated patients (group 1) [p = 0.01; Fig 1 ]. CONCLUSIONS: We conclude that in nonasthmatic, nonseverely ill ambulatory infants with viral bronchiolitis, aerosolized 3% saline solution plus 5 mg terbutaline is effective in decreasing symptoms as compared to 0.9% saline solution plus 5 mg terbutaline.