[Establishment of a predictive model of septic myocardiopathy based on left ventricular global longitudinal strain].

Objectives: To explore the risk factors associated with septic cardiomyopathy and establish a predictive model of the disease based on left ventricular global longitudinal strain (LV GLS). Methods: Data from sepsis patients without a history of cardiac dysfunction who were treated in the Critical Care Department of the Northern Jiangsu People's Hospital from September, 2019 to January, 2021 were included in the analysis. The LV GLS was measured by echocardiography within 72 hours and the patients were divided into a septic myocardiopathy group (LV GLS>-17%) and a normal cardiac function group (LV GLS≤-17%). Clinical data from two groups of patients were collected for univariate analysis. The receiver operating characteristic (ROC) curves of the factors that were statistically different were drawn for exploring the diagnostic and cut-off values. The continuous variable was converted to a dichotomous variable according to the cut-off value. Multivariate logistic regression analysis of sepsis cardiomyopathy was performed to screen the risk factors and create a predictive model. The predictive model was evaluated by ROC curve analysis and the Bootstrap method and shown as a nomograph. Results: Patients in the sepsis cardiomyopathy group had higher levels of high sensitive troponin I (Hs-TnI), procalcitonin (PCT), lactate (Lac), N-terminal pro-brain atriuretic peptide (NT-proBNP), vasopressor dosing intensity (VDI) and sequential organ failure assessment (SOFA) when compared to those in the normal cardiac function group (all P<0.05). The multivariate logistic regression analysis showed that Hs-TnI≥0.131 µg/L (OR=6.71, 95%CI:2.67-16.88, P<0.001), PCT≥40 µg/L (OR=3.08, 95%CI:1.10-8.59, P=0.032), Lac≥4.2 mmol/L (OR=2.80, 95%CI:1.02-7.69, P=0.045), NT-proBNP≥3 270 ng/L (OR=2.67, 95%CI:1.06-6.74, P=0.038) were independent risk factors for septic myocardiopathy. The area under the ROC curve of the predictive model based on the four indexes up-mentioned was 0.838 (95%CI:0.766-0.910), and the C-index was 0.822 (95%CI:0.750-0.894) which indicated the utility of the nomogram. The model had a good predictive ability, accuracy and discrimination. Conclusions: Hs-TnI≥0.131 µg/L, PCT≥40 µg/L, Lac≥4.2 mmol/L and NT-proBNP≥3 270 ng/L are independent risk factors for septic myocardiopathy, and the septic cardiomyopathy predictive model constructed based on these factors has a good diagnostic performance.

Metabolizable and net energy values of corn stored for 3 years for laying hens.

The apparent metabolizable energy (AME), AME corrected to zero-nitrogen retention (AMEn), and net energy (NE) values of 2 corn samples both stored for 3 yr were determined in laying hens with reference diet substitution method. Reference diet was formulated according to standard layer requirement, and test diets contained 50% of corn samples and 50% of the reference diet. Fifty-four Hy-Line Brown hens at the age of 36 wk were used. The heat production and energy metabolism of birds were measured in open-circuit respiratory chambers with 6 replicates (3 birds per replicate) per diet in a randomized design. Birds were fed experimental diets for 7 D in the chamber as adaptation. During the following 3 D, feed intake, metabolizable energy value, nitrogen balance, energy balance, egg production, O2 consumption, CO2 production, and energy efficiency were determined. The AME values of corn 1 and corn 2 were 3,485 and 3,675 kcal/kg DM, respectively. The corresponding AMEn values were 3,452 and 3,596 kcal/kg DM, and the NE values were 2,575 and 2,693 kcal/kg DM, respectively. The NE:AME ratios of corn 1 and corn 2 were 74.4 and 73.3%, respectively. The NE:AMEn ratios of corn 1 and corn 2 were 75.0 and 74.9%, respectively. The AME, AMEn, and NE values of the 2 corn samples both stored for 3 yr were lower than the literature values for fresh corn.

Dual functions of eicosapentaenoic acid-rich microalgae: enrichment of yolk with n-3 polyunsaturated fatty acids and partial replacement for soybean meal in diet of laying hens.

Microalgae (Nannochloropsis sp., NS), with high contents of eicosapentaenoic acid (EPA) and crude protein, may be one of the important n-3 polyunsaturated fatty acid (PUFA) sources and potential protein feed ingredient. The purposes of this study were to enrich yolk with n-3 PUFA by dietary EPA-rich NS supplementation and to evaluate whether it is feasible to partly substitute for soybean meal in laying hens diet. A total of 360 37-wk-old healthy Lohmann Brown laying hens, with similar laying rate and body weight, were randomly allotted to 5 groups (6 replicates, 12 birds/replicate) and fed 5 experimental diets (0, 1, 2, 4, and 8% NS) for 4 wk. The hen performance and egg quality (except yolk color) were not affected (P > 0.05) by the NS supplemental diets. Yolk color score was increased as NS supplementation in diets (P < 0.001), and peaked on about the seventh day in all NS supplemental groups. The concentration of total n-3 PUFA was increased (P < 0.001), while total n-6 PUFA and n-6/n-3 ratio were decreased (P < 0.001) in yolk with increasing NS levels in diets. The 8% NS group had highest docosahexaenoic acid (DHA) and total n-3 PUFA levels, reaching 111.6 mg DHA and 148.6 mg total n-3 PUFA per egg. Maximum DHA, total n-3 PUFA, very long-chain (LC-) n-3 PUFA, and LC-PUFA levels were all observed at day 13 of NS supplementation. In conclusion, dietary NS supplementation enriched yolk with n-3 PUFA (especially DHA) and enhanced yolk color score without adverse effects on performance and egg quality, and indicated the practical feasibility of partial replacement for soybean meal in laying hens diet.

[Effects of dexmedetomidine on myocardium of rats with severe burn at early stage].

Objective: To investigate the effects of dexmedetomidine on myocardium of rats at early stage after severe burn. Methods: Twenty specific pathogen free male SD rats were immersed in 90 ℃ hot water for 20 s, causing 30% total body surface area (TBSA) full-thickness scald (hereafter referred to as burn) on the back. And then they were divided into burn resuscitation group (BR) and burn resuscitation+ dexmedetomidine group (BRD) according to the random number table, with 10 rats in each group. Sodium lactate Ringer's solution (2 mL·kg(-1)·%TBSA(-1)) were intraperitoneally injected into rats of both groups after burn. Dexmedetomidine with dose of 1 µg/kg was intraperitoneally injected into rats of group BRD at the same time point. Another 5 rats in sham injury group (SI) were immersed in 37 ℃ water bath causing sham injury, and fluid resuscitation of rats in group SI was the same as that in group BR. Five rats of group BR and BRD were respectively selected at post burn hour (PBH) 6 and 24. And then left ventricular end-systolic internal diameter (LVIDs), left ventricular end-diastolic internal diameter (LVIDd), ejection fraction (EF), and cardiac output (CO) were determined with small animal ultrasonic imaging system. Plasma levels of cardiac troponin (cTn) I and cTnT were detected by enzyme-linked immunosorbent assay, and morphological changes of myocardium were observed under optical microscope and transmission electron microscope (observed only at PBH 24). In rats of group SI, morphological change of myocardium was observed at PBH 24, and the other indexes were detected as above. Data were processed with one-way analysis of variance and SNK test. Results: At PBH 6, EF value of rats in group BR [(98.0±2.8) %] was obviously higher than that in group SI [(91.0±0.4)%, P<0.05]. The other 3 cardiac ultrasound indexes of rats in group BR were close to those in group SI (with P values above 0.05). Each cardiac ultrasound index of rats between groups BRD and SI was close at PBH 6 (with P values above 0.05). At PBH 24, LVIDs levels of rats in group BR [(0.66±0.59) mm] and group BRD[(0.69±0.27) mm] were obviously lower than LVIDs level of rats in group SI [(1.65±0.33) mm, with P values below 0.05]. LVIDd, EF, and CO levels of rats were close among 3 groups at PBH 24 (with P values above 0.05). At PBH 6, the plasma levels of cTnI [(17.40±1.59) ng/mL] and cTnT [(1 488±229) pg/mL] of rats in group BR were significantly higher than those in group SI [(1.84±0.92) ng/mL and (169±12) pg/mL, with P values below 0.01]. At PBH 6 in group BRD, the plasma level of cTnI of rats [(2.58±0.60) ng/mL] was close to that in group SI (P>0.05), and the plasma level of cTnT [(649±190) pg/mL] was higher than that in group SI (P<0.01). At PBH 24, the plasma levels of cTnI and cTnT of rats in group SI were close to those in groups BR and BRD (with P values above 0.05). At PBH 24, the plasma level of cTnI of rats in group BRD was obviously lower than that in group BR (P<0.01). At PBH 6, the myocardial structures of rats in group BR and group BRD were normal, which were close to myocardial structure of rats in group SI at PBH 24. At PBH 24, obviously damaged myocardial tissue, disorderly arrangement of myofilament, and seriously damaged mitochondria were observed in rats of group BR, which were significantly ameliorated in rats of group BRD. Conclusions: Dexmedetomidine can protect the myocardium of rats with severe burn at early stage.

Effects of dietary energy and lysine levels on growth performance and carcass yields of Pekin ducks from hatch to 21 days of age.

A 2 × 6 factorial experiment, using 2 dietary apparent metabolizable energy (AME) levels (2,750 and 3,050 Kcal/kg) and 6 supplemental lysine (Lys) levels (0, 0.10, 0.20, 0.30, 0.40, and 0.50%), was conducted to study the effects of dietary energy and lysine levels on growth performance and carcass yields of Pekin ducks from hatch to 21 d of age. A total of 576 one-day-old male White Pekin ducks was randomly allotted to 12 dietary treatments, each containing 6 replicate pens with 8 birds per pen. At 21 d of age, body weight gain, feed intake, and feed/gain were measured, and then 2 ducks selected randomly from each pen were slaughtered to evaluate the yields of abdominal fat, breast meat, and leg meat. As a result, birds that were fed basal diets with no Lys supplementation showed growth depression, and significant positive effects of dietary Lys supplementation on body weight gain (P < 0.001), feed intake (P < 0.001), and feed/gain (P = 0.002) were observed as dietary Lys increased gradually among all the groups. In addition, increasing energy levels did not affect overall body weight gain (P > 0.05), but feed intake (P = 0.001) and feed/gain (P = 0.009) decreased significantly between the groups. Dietary Lys levels influenced the yields of breast (P < 0.001) and leg (P = 0.001) meat among all the groups, but dietary energy levels had a significant positive effect only on abdominal fat yield (P = 0.014). The interaction between dietary energy and Lys influenced body weight gain of ducks significantly (P = 0.004). According to the broken-line regression analysis, Lys requirements of Pekin ducks for weight gain at 2,750 and 3,050 Kcal of AME/kg were 0.94 and 0.98%, respectively. It suggested that Lys requirement was higher at 3,050 Kcal of AME/kg than at 2,750 Kcal of AME/kg. Dietary energy content determined feed intake of the ducks, and high-energy diets will require a higher amino acid concentration to compensate for a lower feed intake.

[Comparative study of intratympanic Dexamethasone injection for sudden deafness at different time intervals].

Objective:Comparative study of intratympanic Dexamethasone injection for sudden deafness at different time intervals. Method:One hundred and sixty cases which had been diagnosed sudden deafness were treated by vasodilatortrophic nerve drugs and high pressure oxygen and the same time dexamethasone injective in the middle ear. Injection time intervals were divided into qd, qod, and twice a week. Contral group is not use dexamethasone. Result:The cure rate of qd and qod groups were higher than twice a week group, and the difference was statistically significant (P<0.05). Conclusion:Intratypanic Dexamethasone injection for sudden deafness qd or qod is effective and safe.

Effects of low-protein diets on growth performance and carcass yields of growing French meat quails (France coturnix coturnix).

A dose-response experiment with 5 analyzed dietary crude protein (CP) levels (17.61, 19.73, 21.58, 23.24, and 25.32%) was conducted to investigate the effects of low-protein diets on growth performance and carcass yields of French meat quails from 15 to 42 d of age. All diets were formulated to contain a similar dietary energy level and amino acid profile. A total of 400 fifteen-day-old French quails were divided into 5 experimental treatments and each treatment contained 4 replicate pens of 20 birds (10♂+10♀). At 42 d of age, weight gain, feed intake, CP intake, feed/gain, and the yields of breast part with bone, leg part with bone, and liver of quails from each pen were measured. The results showed significant effects of the low-CP diets on CP intake, weight gain, feed intake, and feed/gain at different experiment periods except for the sixth wk of age (P < 0.05). In addition, as dietary CP decreased from 25.32 to 17.61%, feed intake and feed/gain were increased linearly (P < 0.05), whereas CP intake showed the opposite trend and decreased gradually. On the other hand, the carcass yields of quail were not influenced by reducing dietary CP at 42 day of age (P > 0.05). Based on broken-line regression, 23.0%, 22.5%, and 20.4% were the minimum dietary CP to keep weight gain similar to the quails fed with 25.32% CP diets during the third, fourth, and fifth wk of age, respectively. In summary, with crystalline amino acid supplementation based on a similar amino acid profile, it was possible to formulate the low-protein diets containing about 22.0% CP for growing meat quails without adverse effects on growth and carcass yields of meat quails.

Effects of Dietary Methionine Levels on Choline Requirements of Starter White Pekin Ducks.

A 2×5 factorial experiment, using 2 dietary methionine levels (0.28% and 0.48%) and 5 dietary choline levels (0, 394, 823, 1,239, and 1,743 mg/kg), was conducted to study the effects of dietary methionine status on choline requirements of starter white Pekin ducks from 7 to 28 days of age. Four hundred eighty 7-d-old male White Pekin ducks were randomly allotted to ten dietary treatments, each containing 6 replicate pens with 8 birds per pen. At 28 d of age, weight gain, feed intake, and feed/gain were measured and the legs of all ducks from each pen were examined for incidence of perosis. Perosis and growth depression were observed in choline-deficient ducks and supplementation of choline reduced perosis and significantly increased weight gain and feed intake regardless of dietary methionine levels (p<0.05). In addition, significant positive effects of dietary methionine supplementation on weight gain, feed intake, and feed/gain were observed at any choline level (p<0.05). Supplementation of 1,743 mg/kg choline in diets alleviated the depression of weight gain and feed intake caused by methionine deficiency at 0.28% methionine level. The interaction between choline and methionine influenced weight gain and feed intake of ducks (p<0.05). At 0.28% methionine level, 1,743 mg/kg choline group caused 4.92% and 3.23% amount of improvement in weight gain and feed intake compared with 1,239 mg/kg choline group, respectively. According to the broken-line regression, the choline requirements of starter Pekin ducks for weight gain and feed intake were 1,472 and 1,424 mg/kg at 0.28% methionine level and 946 and 907 mg/kg at 0.48% methionine level, respectively. It suggested the choline recommendations of starter Pekin ducks on a semi-purified diet were 1448 mg/kg at 0.28% methionine level and 927 mg/kg at 0.48% methionine level, respectively. Compared with the adequate methionine level, menthionine deficiency markedly increased the choline requirements of ducks.

Effect of feed consumption levels on growth performance and carcass composition during the force-feeding period in foie gras production of male Mule ducks.

In order to avoid excess feed consumption during the force-feeding period in foie gras production, a dose-response experiment with seven feed consumption levels (450, 540, 630, 720, 810, 900, 990 g/day per bird) was conducted to evaluate the effects of feed consumption levels on growth performance and carcass composition of male Mule ducks from 91 to 102 days of age. One-day-old Mule ducklings (sterile and artificial hybrid of male Albatre Muscovy duck and female Pekin duck were fed a two-phase commercial diets for ad libitum intake from hatching to 91 days of age, followed by graded feeding levels of a corn diet by force-feeding from 91 to 102 days of age. Fifty-six 91-day-old male Mule ducks with similar BW were randomly assigned to seven treatments, with eight birds per treatment. Birds were housed in individual pens. At 102 days of age, final BW was measured and BW gain and feed conversion ratio of ducks from each treatment were calculated from day 91 to 102, and then all ducks were slaughtered to evaluate the yields of skin with subcutaneous fat, abdominal fat, breast meat (including pectoralis major and pectoralis minor), leg meat (including thigh and drum stick), and liver. Significant differences in BW gain, total liver weight and liver relative weight were observed among the treatments (P<0.001). According to the broken-line regression analysis, the optimal feed consumption levels of male Mule ducks from 91 to 102 days of age for maximum BW gain, total liver weight and liver relative weight were 217, 227 and 216 g feed/kg BW0.75·per day, respectively.

Association of human leukocyte antigen DRB1 polymorphism and tuberculosis: a meta-analysis.

OBJECTIVE: To determine whether the human leukocyte antigen DRB1 (HLA-DRB1) is associated with clinical tuberculosis (TB). METHODS: Pooled odds ratios (OR) with 95% confidence intervals (CIs) were used to evaluate the strength of the association between HLA-DRB1 alleles and risk of TB. The χ(2)-based Q-test and I(2) statistics were calculated to examine heterogeneity. Egger's test was performed for the assessment of publication bias. Subgroup analysis was performed based on ethnicity and genotyping methods. RESULTS: A total of 19 case-control studies with 16 alleles (HLA-DRB1*01-HLA-DRB1*16) were included in this meta-analysis. No significant publication bias was detected among these studies. The HLA-DRB1*03 (OR 0.77, 95%CI 0.64-0.93, P = 0.0057) showed a protective effect, while HLA-DRB1*04 (OR 1.24, 95%CI 1.00-1.55, P = 0.0494), HLA-DRB1*08 (OR 1.45, 95%CI 1.14-1.86, P = 0.0030) and HLA-DRB1*16 (OR 1.39, 95%CI 1.04-1.87, P = 0.0269) were significantly associated with increased TB occurrence. Subgroup analysis showed that both ethnicity and genotyping method affected the association between HLA-DRB1*03, HLA-DRB1*04 and HLA-DRB1*08 alleles and TB occurrence. CONCLUSION: These results reinforce the importance of HLA-DRB1 alleles in the development of infectious diseases.

The association of frontal recess anatomy and mucosal disease on the presence of chronic frontal sinusitis: a computed tomographic analysis.

OBJECTIVES: The frontal sinus has the most complex and variable drainage routes of all paranasal sinus regions. The goal of this study was to identify these anatomical factors and inflammation areas relating to chronic frontal sinusitis by comparing radiological presentations in patients with and without frontal sinusitis. METHODS: All adult patients with chronic rhinosinusitis who had received computed tomography (CT) scans of the nasal cavities and paranasal sinuses between October 2010 and September 2011. Logistic regression analysis was used to compare the distribution of various frontal recess cells and surrounding inflammatory conditions in patients with and without frontal sinusitis. RESULTS: Analysis of 240 sides of CT scans was performed with 66 sides excluded. The opacification of the frontal recess and sinus lateralis demonstrated a strong association with an increased presence of frontal sinusitis by multiple logistic regression models. CONCLUSION: Opacification of the frontal recess and sinus lateralis was found to be associated with a significantly increased risk of frontal sinusitis and developing severe blockage of drainage pathways. It provides evidence that mucosal inflammation disease in these two areas is a very important factor leading to chronic frontal sinusitis.

Associations between ethnicity, body composition, and bone mineral density in a Southeast Asian population.

CONTEXT AND OBJECTIVE: Chinese men in Singapore have a higher incidence of hip fractures than Malay and Indian men. We investigated whether there were corresponding ethnic differences in peak bone mineral density (BMD) in young men and whether differences in body composition influenced peak BMD. DESIGN AND SETTING: This was a cross-sectional study of healthy volunteers in a tertiary medical center. PARTICIPANTS: A total of 100 Chinese, 82 Malay, and 80 Indian men aged 21 to 40 years, with body mass index between 18 and 30 kg/m(2) underwent dual-energy x-ray absorptiometry to assess BMD, lean mass (LM) and fat mass (FM), and magnetic resonance imaging to quantify abdominal subcutaneous and visceral adipose tissue. Multiple linear regression models, with adjustment for age and height (as a proxy for skeletal size), were used. RESULTS: Malay and Indian men had significantly higher BMD than Chinese men at the lumbar spine (Malay: B, 0.06 ± 0.02, P = .001; Indian: B, 0.03 ± 0.02, P = .049), femoral neck (Malay: B 0.04 ± 0.02, P = .034; Indian: B, 0.04 ± 0.02, P = .041), hip (Malay: B, 0.05 ± 0.02, P = .016; Indian: B, 0.06 ± 0.02, P = .001), and ultradistal radius (Malay: B, 0.03 ± 0.01, P < .001; Indian: B, 0.02 ± 0.01, P = .029), and this difference was retained after adjustment for LM and FM, except in Malay men at the femoral neck and in Indian men at the ultradistal radius. LM was an important independent determinant of BMD at all sites, whereas FM, subcutaneous adipose tissue, and visceral adipose tissue were not significantly associated with BMD at any site. CONCLUSIONS: Lower peak BMD in Chinese men may partly explain the higher fracture incidence in this ethnic group. Further studies are needed to elucidate the reasons for these ethnic differences in bone accumulation.

Insulin resistance is a major determinant of sustained virological response in genotype 1 chronic hepatitis C patients receiving peginterferon alpha-2b plus ribavirin.

BACKGROUND: Cross-sectional studies suggest insulin resistance is strongly associated with hepatic steatosis and fibrosis in patients with chronic hepatitis C (CHC), which might affect the efficacy of antiviral therapy. Aim To investigate retrospectively the impact of insulin resistance on treatment response in Chinese genotype 1 CHC patients receiving a 24-week course therapy with peginterferon alpha-2b/ribavirin. METHODS: A total of 133 biopsy-proven CHC patients were enrolled for analyses. Insulin resistance was evaluated by homeostasis model assessment of insulin resistance (HOMA-IR). Hepatic fibrosis was graded by the METAVIR scoring system. RESULTS: Mean HOMA-IR progressively elevated along with the severity of hepatic fibrosis (F1-F2 fibrosis: 2.55 +/- 0.16 vs. F3-F4 fibrosis: 3.61 +/- 0.20, P < 0.001). Compared with patients with sustained virological response (SVR), patients without SVR had significantly higher percentages of F3-F4 fibrosis (62.2% vs. 21.6%, P < 0.001) and baseline high viral load (>or=600,000 IU/mL; 64.4% vs. 35.6%, P = 0.038). In addition, patients without SVR had significantly higher plasma levels of insulin (15.03 +/- 0.89 vs. 10.19 +/- 0.55 microU/mL, P < 0.001) and HOMA-IR values (3.76 +/- 0.23 vs. 2.50 +/- 0.15, P < 0.001). Multivariate analyses showed that F1-F2 fibrosis (odds ratio: 4.49, P = 0.001), HOMA-IR < 2 (odds ratio: 7.15, P = 0.005) and pre-treatment hepatitis C virus RNA < 600,000 IU/mL (odds ratio: 3.26, P = 0.012) were the independent factors associated with SVR. CONCLUSIONS: Insulin resistance is a major determinant of SVR in genotype 1 CHC patients receiving peginterferon alpha-2b/ribavirin. Strategies to modify insulin resistance may be effective in enhancing SVR before or during anti-viral therapy.

Electrically assisted magnetic recording in multiferroic nanostructures.

We demonstrate the room-temperature control of magnetization reversal with an electric field in an epitaxial nanostructure consisting of ferrimagnetic nanopillars embedded in a ferroelectric matrix. This was achieved by combining a weak, uniform magnetic field with the switching electric field to selectively switch pillars with only one magnetic configuration. On the basis of these experimental results, we propose to use an electric field to assist magnetic recording in multiferroic systems with high perpendicular magnetic anisotropy.

Strain control of domain-wall stability in epitaxial BiFeO3 (110) films.

We have studied the stability of domains and domain walls in multiferroic BiFeO3 thin films using a combination of piezoelectric force microscopy and phase-field simulations. We have discovered that a film-substrate misfit strain may result in a drastically different thermodynamic stability of two parallel domain walls with the same orientation. A fundamental understanding of the underlying physics, the stress distribution in a domain structure, leads to a novel approach to control the ferroelastic domain stability in the multiferroic BiFeO3 system.

A comparative analysis of the immunological evolution of antibody 28B4.

In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.

Mutational analysis of the affinity maturation of antibody 48G7.

The affinity maturation of antibody 48G7 from its germline predecessor 48G7g has been studied at a molecular level through a combination of structural and biochemical means. Each of the nine somatic mutations accumulated during affinity maturation has been assessed for gain or loss of function in both the germline and affinity-matured antibodies. Individual somatic mutations were found to be either positive or neutral in their effects on affinity for hapten JWJ1, with a marked context-dependence for some sites of mutation. In a number of cases significant cooperativity was found between pairs of somatically mutated residues. Interpretation of the structural changes introduced by many of the point mutations has been possible due to the availability of high-resolution crystal structures of 48G7g and 48G7, and mechanisms by which these structural changes may result in enhanced affinity for hapten have been identified. Precise dissection of structure-function relationships in this system provides additional insights into the role of cooperativity in the evolution of antibody affinity. Comparison of 48G7 with previously characterized systems provides a varied view of the structure-function mechanisms by which the humoral immune system produces large increases in affinity.

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.