In vitro degradation of silk fibroin

A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in 1mg/ml Protease XIV at 37 degrees C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to 12 weeks and (1) prepared for scanning electron microscopy (SEM), (2) lyophilized and weighed, (3) mechanical properties determined using a servohydraulic Instron 8511, (4) dissolved and run on a SDS-PAGE gel, and (5) characterized with Fourier transform infrared spectroscopy. Control samples were incubated in phosphate-buffered saline. Fibroin was shown to proteolytically degrade with predictable rates of change in fibroin diameter, failure strength, cycles to failure, and mass. SEM indicated increasing fragmentation of individual fibroin filaments from protease-digested samples with time of exposure to the enzyme; particulate debris was present within 7 days of incubation. Gel electrophoresis indicated a decreasing amount of the silk 25 kDa light chain and a shift in the molecular weight of the heavy chain with increasing incubation time in protease. Results support that silk is a mechanically robust biomaterial with predictable long-term degradation characteristics.     

Dual function of Langerhans cells in skin TSLP-promoted TFH differentiation in mouse atopic dermatitis

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Frequency and properties of naturally occurring adherent piliated strains of Haemophilus influenzae type b.

We found that 41 of 75 (55%) children with Haemophilus influenzae type b disease (70 cases of meningitis, 2 of cellulitis, 2 of septic arthritis, and 1 of epiglottitis) and 2 of 120 (1.7%) children with upper respiratory infection were colonized with H. influenzae type b in the nasopharynx (NP). Of these 43 NP strains from children with systemic H. influenzae type b disease, 7 (16%) adhered to human buccal epithelial cells. The strains isolated from the systemic site of all children, including children from whose NP adherent bacteria were isolated, did not adhere to buccal epithelial cells in vitro. Each adherent NP strain had biotype (I), serotype (b), and antibiotic susceptibility (sensitive) similar to that of the corresponding nonadherent systemic isolate. With one exception, all NP-systemic pairs had similar major outer membrane proteins. Six of the seven NP strains had a protein band in the whole cell lysate preparation with a molecular weight between 22,000 and 23,000, which could not be seen in the nonadherent cerebrospinal fluid strains. Electron micrographs of all adherent strains showed that more than 95% of the organisms examined were highly piliated, whereas the nonadherent strains were not piliated. All piliated strains agglutinated human erythrocytes. Adherence to buccal epithelial cells and agglutination of erythrocytes could not be blocked by mannose or alpha-methyl-D-mannoside. We speculate that piliation is not important for NP colonization by H. influenzae type b and that the loss of pili may be required for host invasion.     

Anti-pyruvate dehydrogenase autoantibodies in primary biliary cirrhosis

Antimitochondrial antibodies (AMA) may be detected in 95% of patients with primary biliary cirrhosis (PBC). The target autoantigens for the AMA were recently identified as four closely related metabolic enzymes located in the mitochondria. We have purified the pyruvate dehydrogenase (PDH) enzyme from bovine heart, showing that all PBC sera reacted with a 74-kd band. PDH was utilized to establish an ELISA assay for detecting the relevant antibodies. One hundred twelve of 120 sera from patients with PBC (95%) reacted with the PDH but none of the 201 control sera, including normal subjects and a panel of sera from other patients with liver diseases, showed similar reactivity. In 77% of the PBC sera the anti-PDH antibody isotype was identified as a combination of IgG and IgM, while in 18% only IgM was detected. In 5% of the sera the isotype was confined to IgG. PBC sera specifically inhibited the PDH enzyme activity. The enzyme inhibition correlated with the anti-PDH antibody titers. Thus, PDH seems to be one of the major target epitopes for AMA observed in sera of patients with PBC.     

Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP)

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur in lymphoblastoid cells and in lesional cells of patients with FOP, mutations have not been identified in the BMP4 gene, suggesting that the mutation in FOP may reside in a BMP4-interacting factor or in another component of the BMP4 pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A recent case report described a heterozygous 42-bp deletion in the protein-coding region of the noggin gene in a patient with FOP. In order to determine if noggin mutations are a widespread finding in FOP, we examined 31 families with 1 or more FOP patients. Linkage analysis with an array of highly polymorphic microsatellite markers closely linked to the noggin gene was performed in four classically-affected multigenerational FOP families and excluded linkage of the noggin locus to FOP (the multipoint lod score was -2 or less throughout the entire range of markers). We sequenced the noggin gene in affected members of all four families, as well as in 18 patients with sporadic FOP, and failed to detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of 4 of these patients plus an additional 9 patients also failed to reveal any mutations. Among the samples analyzed by SSCP and DNA sequencing was an independently obtained DNA sample from the identical FOP patient previously described with the 42-bp noggin deletion; no mutation was detected. Examination of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate the possibility of a somatic mutation in the noggin gene which could be carried by a small subset of white blood cells, also failed to detect the presence of the reported 42-bp deletion. We conclude that mutations in the coding region of noggin are not associated with FOP.     

New families of adhesion molecules play a vital role in platelet functions

Adhesion molecules play a crucial part in cell-matrix and in cell-cell interactions. These interactions, which are essential to the body's defense processes, involve adhesion molecules belonging to different families: integrins, immunoglobulins and selectins. Integrins are expressed by a large number of tissues, whereas other adhesion molecule families are restricted to a small number of cell types. A recent symposium dealt with the recruitment of circulating platelets at specific sites, their adhesion to extracellular matrix components and their activation by agonists leading to aggregation or attachment to other cells. These events, supporting hemostasis and thrombosis, involve integrins, selectins and other adhesion molecules. This report focuses on newly reported integrins (GPIa, GPIc, GPIIa), selectins (GMP-140) and GPIIIb, previously known as 'minor' surface oriented platelet glycoproteins. Major membrane glycoproteins such as GPIIb-IIIa (an integrin) and GPIb, which also play a vital role in platelet functions, have been extensively reviewed elsewhere.     

Phospholipid metabolism of herpesvirus-infected and uninfected rabbit kidney cells

The incorporation of labeled precursors into the phospholipids of uninfected rabbit kidney cells in stationary phase was studied with the following results: (1) Incorporation of ³²P into cytoplasmic phospholipids is rapid and reaches a plateau by 40 hr of incubation with the isotope; incorporation into the nuclear fraction, however, continues to increase linearly. (2) The different phospholipids become labeled at different rates. (3) The inner nuclear membrane fraction has a different phospholipid composition from the outer nuclear or the cytoplasmic fractions; it contains a significantly larger amount of sphingomyelin.     

Genetic susceptibility and anti-human platelet antigen 5b alloimmunization role of HLA class II and TAP genes

Platelet alloimmunization may result in post-transfusion purpura, and during pregnancy may cause neonatal alloimmune thrombocytopenia (NAIT), with a frequency estimated at 1.3 per 1000 live births. The risk of morbidity is significant: 20% of affected infants have neurologic sequelae and the death rate is about 10%. A better understanding of the immune response to platelet alloantigens would allow for a better definition, and thus better management of pregnant women at high risk. Limited data are available on the immune response against HPA-5b, the second most frequent antigen, after HPA-la, implicated in NAIT. We studied HLA class II and TAP gene polymorphism in 50 women immunized against HPA-5 system antigens. Our results suggest a strong association of alloimmunization with a cluster of HLA DR molecules sharing a particular polymorphic amino acid sequence at position 69–70 (Glu-Asp encoded by GAA-GAC nucleotide sequence) of the DRβl chain (RR = 2.95, RR = 5.70 when patients were homozygous for this sequence), and a negative association with the DRB1^*0301 allele (2.1% vs. 28%; RR = 0.08). Furthermore, increased frequency of a TAP2 dimorphism at position 379 was observed in immunized women against the HPA-5 antigens (RR = 4.7).     

Phenotype-genotype correlations in X linked retinitis pigmentosa.

Retinitis pigmentosa (RP) represents a group of clinically heterogeneous retinal degenerations in which all modes of inheritance have been described. We have previously found two different clinical profiles in X linked RP as a function of age and mode of onset. The first clinical form has very early onset with severe myopia. The second form starts later with night blindness with mild myopia or none. At least two genes have been identified in X linked forms, namely RP2 (linked to DXS7, DXS255, and DXS14) and RP3 (linked to DXS84 and OTC) on the short arm of the X chromosome. In order to contribute to phenotype-genotype correlations in X linked RP, we tested the hypothesis that the two clinical profiles could be accounted for by the two different gene loci. The present study provides evidence for linkage of the clinical form with early myopia as the onset symptom with the RP2 gene (pairwise linkage to DXS255: Z = 3.13 at theta = 0), while the clinical form with later night blindness as the onset symptom is linked to the RP3 gene (pairwise linkage to OTC: Z = 4.16 at theta = 0).     

Myosin VIIA gene: heterogeneity of the mutations responsible for Usher syndrome type IB

Usher syndrome is recognized as the most frequent cause of hereditary deaf-blindness. Usher syndrome type I (USH1), the most severe form of the disease, is characterized by profound congenital sensorineural deafness, constant vestibular dysfunction, and retinitis pigmentosa of prepubertal onset. This form is genetically heterogeneous and five loci (USH1A-E) have been mapped thusfar. However, only the gene responsible for USH1 B (which accounts for approximately 75% of USH1 cases) has been characterized. It encodes a long-tailed unconventional myosin, myosin VIIA, with a predicted 2215 amino acid sequence. Primers covering the complete myosin VIIA coding sequence as well as the 3' non coding sequence were designed, allowing direct sequence analysis of each of the 48 coding exons and flanking splice sites in seven patients affected by USH1. Four novel mutations were thereby identified. The possibility should now be considered of a sequence-based prenatal diagnosis in some of the families affected by this very severe form of Usher syndrome.