Enzymatic mineralization generates ultrastiff and tough hydrogels with tunable mechanics.

The cartilage and skin of animals, which are made up of more than fifty per cent water, are rather stiff (having elastic moduli of up to 100 megapascals) as well as tough and hard to break (with fracture energies of up to 9,000 joules per square metre). Such features make these biological materials mechanically superior to existing synthetic hydrogels. Lately, progress has been made in synthesizing tough hydrogels, with double-network hydrogels achieving the toughness of skin and inorganic-organic composites showing even better performance. However, these materials owe their toughness to high stretchability; in terms of stiffness, synthetic hydrogels cannot compete with their natural counterparts, with the best examples having elastic moduli of just 10 megapascals or less. Previously, we described the enzyme-induced precipitation and crystallization of hydrogels containing calcium carbonate, but the resulting materials were brittle. Here we report the enzyme-induced formation of amorphous calcium phosphate nanostructures that are homogenously distributed within polymer hydrogels. Our best materials have fracture energies of 1,300 joules per square metre even in their fully water-swollen state-a value superior to that of most known water-swollen synthetic materials. We are also able to modulate their stiffness up to 440 megapascals, well beyond that of cartilage and skin. Furthermore, the highly filled composite materials can be designed to be optically transparent and to retain most of their stretchability even when notched. We show that percolation drives the mechanical properties, particularly the high stiffness, of our uniformly mineralized hydrogels.

Interleukin-6 concentrations in the urine and dipstick analyses were related to bacteriuria but not symptoms in the elderly: a cross sectional study of 421 nursing home residents.

BACKGROUND: Up to half the residents of nursing homes for the elderly have asymptomatic bacteriuria (ABU), which should not be treated with antibiotics. A complementary test to discriminate between symptomatic urinary tract infections (UTI) and ABU is needed, as diagnostic uncertainty is likely to generate significant antibiotic overtreatment. Previous studies indicate that Interleukin-6 (IL-6) in the urine might be suitable as such a test. The aim of this study was to investigate the association between laboratory findings of bacteriuria, IL-6 in the urine, dipstick urinalysis and newly onset symptoms among residents of nursing homes. METHODS: In this cross sectional study, voided urine specimens for culture, urine dipstick and IL-6 analyses were collected from all residents capable of providing a voided urine sample, regardless of the presence of symptoms. Urine specimens and symptom forms were provided from 421 residents of 22 nursing homes. The following new or increased nonspecific symptoms occurring during the previous month were registered; fatigue, restlessness, confusion, aggressiveness, loss of appetite, frequent falls and not being herself/himself, as well as symptoms from the urinary tract; dysuria, urinary urgency and frequency. RESULTS: Recent onset of nonspecific symptoms was common among elderly residents of nursing homes (85/421). Urine cultures were positive in 32% (135/421), Escherichia coli was by far the most common bacterial finding. Residents without nonspecific symptoms had positive urine cultures as often as those with nonspecific symptoms with a duration of up to one month. Residents with positive urine cultures had higher concentrations of IL-6 in the urine (p < 0.001). However, among residents with positive urine cultures there were no differences in IL-6 concentrations or dipstick findings between those with or without nonspecific symptoms. CONCLUSIONS: Nonspecific symptoms among elderly residents of nursing homes are unlikely to be caused by bacteria in the urine. This study could not establish any clinical value of using dipstick urinalysis or IL-6 in the urine to verify if bacteriuria was linked to nonspecific symptoms.

Association of soluble CD89 levels with disease progression but not susceptibility in IgA nephropathy.

The Fc-α receptor (FcαR/CD89) is involved in IgA complex formation and may affect the development of IgA nephropathy (IgAN). In this study, we tested the genetic variations of the CD89 gene in relation to disease susceptibility in IgAN and the expression of soluble CD89 (sCD89) in sera of patients with IgAN and in controls. There was a significant difference between the levels of sCD89-IgA complexes, measured by sandwich enzyme-linked immunosorbent assay (ELISA), in 177 patients with IgAN with and without disease progression at the time of first diagnosis. No such difference was found in 42 patients with other renal diseases. The patients with IgAN without disease progression had stable but high levels of sCD89 over 5-15 years of follow-up in contrast to stable but low levels of sCD89 in the disease progression group. Moreover, levels of sCD89 complexes were correlated with one of the five CD89 genetic variants in 212 patients with IgAN and 477 healthy Caucasians; the single-nucleotide polymorphism (SNP) rs11084377 was significantly associated with a lower expression of sCD89. However, no association between CD89 gene polymorphisms and susceptibility to IgAN was detected. Thus, we found an association between the levels of sCD89-IgA complexes in serum and the severity of IgAN, and a possible genetic component in regulating the production or expression of sCD89.

Genetic variation in the transforming growth factor-beta1 gene is associated with susceptibility to IgA nephropathy.

BACKGROUND: There is growing evidence of genetic risk for susceptibility to IgA nephropathy. Among several candidate genes related to immunological regulation in renal tissue, TGFB1 is known to be a contributor to proliferation and the development of fibrosis. METHODS: We analysed several SNPs in a region of this gene using 212 DNA samples from biopsy-proven IgA nephropathy patients, 146 men and 66 women and 477 healthy age-matched controls (321 men and 156 women) from the same population in Sweden. RESULTS: Frequencies of four out of five selected SNPs (rs6957, rs2241715, rs1800471, rs1982073 and rs1800469) were found to significantly differ between male patients and male controls in a co-dominant model (corrected P

Urinary proteins in children with urinary tract infection.

The aim of this study was to test our hypothesis that the urinary excretion of C-reactive protein (CRP), alpha 1-microglobulin (A1M), retinol-binding protein (RBP) and Clara cell protein (CC16) is increased in children with urinary tract infection (UTI) and relates to renal damage as measured by acute dimercaptosuccinic acid (DMSA) scintigraphy. Fifty-two children <2 years of age with UTI were enrolled in the study, 44 of whom were febrile. The control group consisted of 23 patients with non-UTI infection and elevated serum CRP (s-CRP) levels. Thirty-six patients had abnormal DMSA uptake, classified as mild, moderate or severe damage (DMSA class 1, 2, 3, respectively). There was a significant association between DMSA class and the excretion of urinary RBP (u-RBP) and u-CC16. There was also a significant difference in u-CRP levels between children with UTI and control children with non-UTI infections, although u-CRP excretion was not significantly correlated to DMSA class. In conclusion, the urinary excretion of the low-molecular-weight proteins RBP and CC16 showed a strong association with uptake defects on renal DMSA scans. The urinary level of CRP seems to distinguish between children with UTI and other febrile conditions. A combination of these biomarkers may be useful in the clinical assessment of children with UTI.

Common variations in the IL4R gene affect splicing and influence natural expression of the soluble isoform.

We previously found the soluble interleukin 4 receptor (sIL4R) to be differently expressed in allergic asthma patients compared to healthy individuals. Here we present data demonstrating the involvement of the sequence variations, c.912-1003A > G, c.912-833T > C, c. 912-630A > G, and c.912-577A > G, in the expressional regulation of IL4R splice variants. By using an IL4R minigene construct, genomic DNA and mRNA from asthma patients and nonasthmatic individuals, we analyzed the function of four highly-linked SNPs, flanking the alternatively-spliced exon in the IL4R gene. Results from the minigene assay showed that the form containing the minor alleles significantly decreased the expression of the soluble IL4R (exon 8+) variant, a decrease that could only be seen in the major construct after increasing amounts of either the splicing factor SRp20, or YT521-B. Analysis of mRNA expression in our human material confirmed the results, demonstrating lower expression of the sIL4R in patients and controls carrying the minor alleles. Together these results show sequence variations as a possible way of altering alternative splicing selection of IL4R in vivo.

Impaired kidney graft survival is associated with the TNF-alpha genotype.

BACKGROUND: The TNF2 allele at position -308 of the tumor necrosis factor (TNF)-alpha gene is associated with high TNF production. The purpose was to study the association of this gene polymorphism with rejection episodes and graft survival after kidney transplantation. METHODS: A retrospective analysis of transplant outcomes of patients who only had been treated with one single form of immunosuppression consisting of cyclosporine, azathioprine, and prednisolon was performed. RESULTS: We found that 115 (73%) patients had the TNF1/TNF1 genotype, whereas 42 (27%) were TNF2 positive. There was no difference in the overall acute rejection frequency between these two groups (50% in each), but our data showed a non-significant tendency towards a higher frequency of steroid resistant rejections in the TNF2 positive group (57% vs. 40%). There was no significant difference in graft survival between the two genotype groups, although an early tendency towards worse survival was seen in TNF2 recipients. However, the TNF2 positive recipients with rejection episodes had far worse graft survival compared with the TNF1/TNF1 recipients with rejection episodes (P<0.02). No difference was seen between the two genotype groups in patients without rejection episodes. CONCLUSION: Our data propose that potentially high TNF producers with the TNF2 allele do not have an increased risk for rejection episodes, but if rejection episodes occur, they have a significantly increased risk for early graft loss. TNF production may intensify rejection, but is not a primary factor for the induction of such acute immune activation.

Long term enhancement of the IgG2 antibody response to Haemophilus influenzae type b by breast-feeding.

SUBJECTS: Sets of sera were obtained from 30 children <6 years of age with invasive type b (Hib) infection and their mothers. Duration and mode of breast-feeding were monitored. Titers of IgG1, IgG2, IgA and IgM antibodies against Hib capsular polysaccharide were determined in sera taken during the acute illness and during early and late convalescence. RESULTS: Children 18 months or older with longer durations of exclusive breast-feeding (13 weeks or more; mean, 19.3 weeks) had higher Hib antibody concentrations of the IgG1, IgG2, IgA and IgM isotypes than those with a shorter duration of exclusive breast-feeding (<13 weeks; mean, 5.4 weeks). The difference was greatest for the IgG2 isotype. In regression analyses the association between the duration of exclusive breast-feeding and the anti-Hib IgG2 concentration was significant when breast-feeding, type of Hib infection, maternal Hib antibody titer and age were used as explanatory factors. In the group of 14 children <18 months of age no significant differences were noted. DISCUSSION: This study indicates the presence of a long lasting enhancing effect of breast-feeding on the antibody response to Hib in children, in particular on IgG2 Hib antibody production. This may result from the content in the milk of IFN-gamma and IFN-gamma-producing cells and possibly other factors, which can support IgG2 antibody production.

Glial cell line-derived neurotrophic factor is increased in cerebrospinal fluid but decreased in blood during long-term pain.

Glial cell line-derived neurotrophic factor (GDNF) is involved in inflammation and pain, roles which remain to be delineated clinically. We aimed to evaluate the role of central nervous and peripheral GDNF in long-term pain patients and in controls by analysing intrathecal and blood concentrations of GDNF. Simultaneous measurements of pro-inflammatory cytokines IL-1beta, TNF-alpha and IL-6, anti-inflammatory cytokine IL-10 and chemokine IL-8 served to define inflammatory responses. Generally, blood levels of GDNF were higher than corresponding intrathecal levels. Pain was associated with levels of GDNF that were increased intrathecally, but decreased in blood. IL-8 was uniformly higher in pain patients.

Long-term treatment results and the immunoglobulin G subclass distribution patterns of proteinase-3-antineutrophil cytoplasm antibody (ANCA) and myeloperoxidase-ANCA in ANCA-associated vasculitis.

OBJECTIVE: Small vessel vasculitis associated with antibodies to neutrophil cytoplasm antigens has been denominated antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV). MATERIAL AND METHODS: Ninety-eight patients with various forms of AAV with renal involvement were studied retrospectively with regard to treatment, side-effects and outcome. The immunoglobulin G (IgG) subclass distribution patterns in serum were determined in 51 patients with nephelometry and those of anti-proteinase-3 (PR3) and anti-myeloperoxidase (MPO) in 44 patients by enzyme-linked immunosorbent assay. RESULTS: Fifty-nine patients with a mean age of 63 years were given treatment with intermittent intravenous regimens of cyclophosphamide and continuous corticosteroids, whereas 39 patients with a mean age of 58 years were given continuous oral treatment. Malignancy, mainly due to skin tumours, was more common in AAV than in the general population. The total IgG subclass distribution pattern was asymmetric. The response to PR3 was of IgG(1), IgG(3) and IgG(4) isotypes, while IgG(1) and IgG(3) predominated in the response to MPO. CONCLUSION: The aberrant IgG subclass distribution pattern detected in the autoantibodies may be of importance in the pathogenesis of AAV.

A module-based analytical strategy to identify novel disease-associated genes shows an inhibitory role for interleukin 7 Receptor in allergic inflammation.

BACKGROUND: The identification of novel genes by high-throughput studies of complex diseases is complicated by the large number of potential genes. However, since disease-associated genes tend to interact, one solution is to arrange them in modules based on co-expression data and known gene interactions. The hypothesis of this study was that such a module could be a) found and validated in allergic disease and b) used to find and validate one ore more novel disease-associated genes. RESULTS: To test these hypotheses integrated analysis of a large number of gene expression microarray experiments from different forms of allergy was performed. This led to the identification of an experimentally validated reference gene that was used to construct a module of co-expressed and interacting genes. This module was validated in an independent material, by replicating the expression changes in allergen-challenged CD4+ cells. Moreover, the changes were reversed following treatment with corticosteroids. The module contained several novel disease-associated genes, of which the one with the highest number of interactions with known disease genes, IL7R, was selected for further validation. The expression levels of IL7R in allergen challenged CD4+ cells decreased following challenge but increased after treatment. This suggested an inhibitory role, which was confirmed by functional studies. CONCLUSION: We propose that a module-based analytical strategy is generally applicable to find novel genes in complex diseases.

Revaccination does not improve an observed deficit in antibody responses in Pakistani adults born of a lower birth weight.

We have previously shown that the generation of antibodies to a polysaccharide vaccine (Typhim Vi) is compromised in Pakistani adults born of a lower birth weight. To assess whether this represents a true B-cell-dependent deficit, we revaccinated subjects with a second dose of the same vaccine and with a polysaccharide-protein conjugate vaccine to a different polysaccharide antigen (conjugated Haemophilus influenzae type b (Hib) vaccine). Anti-Vi IgG levels remained positively correlated with birth weight (p=0.0284) but no associations were observed between anti-Hib IgG levels and size at birth. These findings indicate that small size at birth results in a poor antibody response to vaccination with a polysaccharide antigen vaccine in adulthood, even following a second dose of the vaccine. No such association was observed in response to a polysaccharide-protein conjugate vaccine indicating an early-life programming effect on the generation of antibodies during a B-cell-dependent immune response.

Avidity of tetanus and Hib antibodies after childhood acute lymphoblastic leukaemia - implications for vaccination strategies.

AIM: To investigate the possible relationship between serum levels and avidities of antibodies against tetanus toxoid (TT) and Haemophilus influenzae type b (Hib) in children that were vaccinated after treatment for childhood acute lymphoblastic leukaemia (ALL). METHODS: The study groups were 31 paediatric patients with ALL and 18 healthy controls. All subjects were vaccinated with TT and a protein conjugated Hib vaccine. Antibody levels were analysed at three time points: At diagnosis of ALL, after cessation of treatment before vaccination and three weeks after vaccination. Avidity was measured twice, with a thiocyanate elution assay, at diagnosis of ALL and three weeks after vaccination. RESULTS: There was a correlation between level and avidity of tetanus antibodies after vaccination (r(s) = 0.59, P < 0.001). In the standard-risk and intermediate-risk ALL groups, all patients responded with protective levels of tetanus antibodies with normal avidity. In the high-risk ALL group 7/9 patients had subprotective levels of tetanus antibodies after vaccination and concomitantly the lowest avidity, implying poor protection against tetanus. No patients were found with low levels and low avidity of anti-Hib IgG, and 29/31 patients had full protection after a single dose of conjugated Hib-vaccine. CONCLUSION: The vaccination strategy after childhood ALL must be different for low-risk and high-risk ALL groups, since the high-risk group fail to elicit a recall response to tetanus.

Interferon-gamma dose-dependently inhibits prostaglandin E2-mediated dendritic-cell-migration towards secondary lymphoid organ chemokines.

Monocyte-derived human dendritic cells (MoDCs) are increasingly applied as cellular vaccines for cancer patients. Important features for their efficacy include high migratory responsiveness to lymph node-chemokines and most likely their ability to produce bioactive IL-12 p70 upon subsequent contact with CD40 ligand-expressing T-cells. The current standard DC-maturation cocktail for clinical trials is inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) combined with prostaglandin E(2) (PGE(2)), inducing phenotypically mature MoDCs with high migratory responsiveness to CCR7 ligands. This cocktail does not, however, induce or prime for production of IL-12 p70. Addition of IFN-gamma to PGE(2)-containing maturation cocktails has been shown to prime for substantial production of IL-12 p70 by subsequent CD40 ligation, but the impact of IFN-gamma on phenotypic maturation and migratory responsiveness induced by PGE(2)-containing inflammatory stimuli still remains elusive. Here, we demonstrate that addition of IFN-gamma to the standard maturation cocktail decreased CCR7 mRNA and down-regulated CCR7 expression on MoDCs in a dose-dependent manner. Moreover, addition of IFN-gamma was found to suppress MoDC-migration towards the CCR7-ligands CCL19 and CCL21. These novel findings indicate that addition of IFN-gamma to DC-maturation stimuli may have no beneficial impact on MoDC-vaccine efficiency and further implicate IFN-gamma as a negative feedback factor in DC migration towards draining lymph nodes when full-blown Th1-type responses are established. Such mechanism may restrict an uncontrolled and potentially harmful amplification of the adaptive Th1 response.

Cytokines in the placenta of Pakistani newborns with and without intrauterine growth retardation.

Although intrauterine growth retardation (IUGR) is a major risk factor for increased neonatal mortality and morbidity, the mechanisms behind it are not clear. We analyzed cytokine gene expression and gene polymorphisms in infants with and without IUGR in Pakistan, where IUGR is very common. 45 IUGR and 55 control mother/infant pairs were studied. mRNA for IL-10, IL-8, TNF-alpha, TGF-beta, IL-6, IL-4, IL-1beta, IL-12, IFN-gamma and GAPDH was quantified with RT-PCR from placenta. Cytokine and cytokine receptor gene polymorphisms for -1087IL10, -308TNFA, -174IL6, +915TGFB1, intron 2 IL1RN, +36TNFR1, 150V IL4RA and -159CD14 were determined from genomic DNA. The serum levels of IL-1beta, IL-6, IL-8, IL-10, IL-12, TNF-alpha and TGF-beta were measured. There was a significant decrease of IL-10 and IL-12, but increase of TGF-beta in the decidua and similarly decrease of IL-10, but increase of TGF-beta in the trophoblasts of the IUGR placentas compared with the non-IUGR placentas. We found significantly lower levels of IL-1beta in serum from the mothers of the IUGR infants and of TGF-beta in serum of the infants with IUGR compared with the non-IUGR infants. We note that the IL-10 mRNA expression in the decidua was down-regulated, but the TGF-beta mRNA up-regulated in IUGR placentas of mothers from a population with multiple risk factors for IUGR. We propose that the low IL-10 in the placenta may be involved in the pathogenesis of IUGR and might possibly be treatable.

Association of the -159 CD14 gene polymorphism and lack of association of the -308 TNFA and Q551R IL-4RA polymorphisms with severe chronic periodontitis in Swedish Caucasians.

BACKGROUND: Severe forms of periodontitis are suggested to have a genetic basis. OBJECTIVE: The aim of the present investigation was to study the association of gene polymorphisms related to some immune regulation components (G-308A TNFA, Q551R IL-4RA and C-159T CD14) with severe chronic periodontitis. MATERIALS AND METHODS: Sixty patients (aged 36-74 years; mean 54.5+/-8.5) with severe and generalized chronic periodontitis were included. The patients exhibited bone loss >50% at all teeth. Thirty-nine periodontally healthy subjects between 35 and 78 years of age (mean 51.0+/-10.9) were recruited as controls. DNA was isolated from peripheral blood cells and genotyping was performed by combination of PCR and restriction endonuclease mapping. RESULTS: While gene polymorphisms for TNFA and IL-4RA did not show any association with severe chronic periodontitis, the analysis of the -159 CD14 gene polymorphism revealed significant differences between test and control groups. The proportion of subjects that exhibited the TT genotype was significantly smaller in the group with severe periodontitis than in periodontal healthy group (p=0.028; Fisher's exact test). The C allele carriage was 90% in the periodontitis group and significantly higher than in the healthy control group (72%). CONCLUSION: It is suggested that the -159 CD14 gene polymorphism is associated with chronic periodontitis in Caucasian subjects of a north European origin.

Intensive treatment for childhood acute lymphoblastic leukemia reduces immune responses to diphtheria, tetanus, and Haemophilus influenzae type b.

OBJECTIVES: Immunity to diphtheria toxoid (D), tetanus toxoid (T), and Haemophilus influenzae type b (Hib) is affected in children with acute lymphoblastic leukemia (ALL). The aims were to examine immunity and to compare the response to immunization at 1 or 6 months after treatment. METHODS: Thirty-one patients were immunized with DT and conjugated Hib vaccine (ActHib) at 1 month or 6 months after treatment of ALL with the NOPHO 92 protocol. Antibody levels were determined before and 3 weeks after vaccination. Specific T and Hib antibody-secreting cells of IgG/IgA/IgM isotypes were analyzed in peripheral blood using an ELISPOT technique. RESULTS: All specific antibody levels decreased during ALL treatment, and protective levels after treatment were noted for 17% against D, 33% against T, and 100% against Hib. No high-risk patient had full D or T protection after treatment. After vaccination all the standard- and intermediate-risk patients achieved full protection against D, T, and Hib. The high-risk group showed insufficient immune response (full protection after vaccination: D 56%, T 22%, Hib 78%). No difference was found between vaccination at 1 month or 6 months after treatment. The poor antibody production in the high-risk group correlated to low numbers of antibody-secreting cells. CONCLUSIONS: Nonprotective antibody levels against D, T, and Hib after childhood ALL are more common than previously thought. Insufficient immune response was restricted to the high-risk group and was related to a low number of memory B cells in this study. Immunizations should be included in follow-up after childhood ALL, and the policy should be adapted to treatment intensity.

Association of the -1087 IL 10 gene polymorphism with severe chronic periodontitis in Swedish Caucasians.

BACKGROUND: Severe forms of periodontitis are suggested to have a genetic basis. OBJECTIVE: The aim of the present investigation was to study association of an IL10 gene polymorphism (G to A transition at the -1087 position) with severe chronic periodontitis. MATERIALS AND METHODS: Two groups of Swedish Caucasian subjects were included. One group consisted of 60 patients (aged 36-74 years; mean 54.5+/-8.5) with severe and generalized chronic periodontitis. The patients exhibited bone loss >50% at all teeth. Thirty-nine periodontally healthy subjects between 35-78 years of age (mean 51.0+/-10.9) were also recruited. DNA was isolated from peripheral blood cells and genotyping was performed by combination of PCR with restriction endonuclease mapping. RESULTS: The proportion of subjects that exhibited the GG genotype was significantly larger in the group with severe periodontitis than in the periodontally healthy group. The difference regarding the occurrence of the GG genotype between the two groups was more conspicuous in non-smokers and yielded an odds ratio of 6.1. The G allele carriage in non-smokers was >90 % in the periodontitis group and was significantly higher than in the healthy controls. CONCLUSION: It is suggested that the -1087 IL10 polymorphism in Caucasian subjects of a north European origin is associated with severe chronic periodontitis.

Aberrations in placental cytokine mRNA related to intrauterine growth retardation.

During normal pregnancy, a predominance of Th2 type cytokines prevails and is considered to protect the fetus. Animal experiments suggest that an increase of Th1 type cytokines may instead have deleterious effects. We have studied with the reverse transcription PCR technique mRNA for IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, transforming growth factor-beta, tumor necrosis factor-alpha, and interferon-gamma in placentas from full-term appropriately grown newborns, newborns with intrauterine growth retardation (IUGR) and newborns who were only small for gestational age. The mRNA for IL-10 was significantly reduced in the IUGR placentas (p < 0.05), whereas the mRNA for IL-8 was significantly higher (p < 0.05) for the IUGR cases compared with the full-term neonates. It might be that reduced IL-10 in the placenta is involved in the pathogenesis of IUGR.