Chlamydia pneumoniae (TWAR).

Chlamydia pneumoniae (TWAR) is a recently recognized third species of the genus Chlamydia that causes acute respiratory disease. It is distinct from the other two chlamydial species that infect humans, C. trachomatis and C. psittaci, in elementary body morphology and shares less than 10% of the DNA homology with those species. The organism has a global distribution, with infection most common among children between the ages of 5 and 14 years. In children, TWAR infection is usually mild or asymptomatic, but it may be more severe in adults. Pneumonia and bronchitis are the most common clinical manifestations of infection, and TWAR is responsible for approximately 10% of cases of pneumonia and 5% of cases of bronchitis in the United States. The microimmunofluorescence serologic assay is specific for TWAR and can distinguish between recent and past infections. The organism can be isolated in cell culture; however, PCR techniques have recently facilitated its detection in tissues and clinical specimens.     

Screening for cognitive impairment in older adults attending an eye clinic

PURPOSE: We conducted a cross-sectional study examining potentially modifiable factors associated with cognitive impairments (mild or severe) in older whites, African Americans and Hispanics attending an outpatient eye clinic. METHODS: In-clinic interviews and physical examinations assessed social, demographic and health information from 100 consecutive Hispanic, African-American and white adults aged > or = 55. Our primary outcome was presence of any cognitive impairment (mild or severe) using the St. Louis University Mental Status Examination (SLUMS) scale. RESULTS: Of the 100 subjects, 65 screened positive for cognitive impairments on the SLUMS cognitive instrument: 46 with mild cognitive impairment and 19 with severe impairment (possible dementia). African-American and Hispanic adults (nonwhites) were significantly more likely to have cognitive impairment compared to white adults (OR 2.80: 95% CI = 1.05-7.44), independent of age, years of education and systolic blood pressure. Subjects with diabetes also had increased odds of cognitive impairments (OR 3.28, 95% CI = 1.21-8.90) even after adjusting for relevant confounders. There was a nonsignificant trend between visual acuity impairment and cognitive impairment (p = 0.059). CONCLUSIONS: Sixty-five percent of adults aged > or = 55 attending the eye clinic screened positive for cognitive impairments, with higher rates among nonwhites and adults living with diabetes.     

Effect of Group A Streptococcal Cysteine Protease on Invasion of Epithelial Cells

Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.     

An extensive study of human IgE cross-reactivity of Blo t 5 and Der p 5

BACKGROUND: Dual sensitization by Blomia tropicalis and Dermatophagoides pteronyssinus mites is common in tropical and subtropical countries. The human IgE cross-reactivity between clinical important group 5 allergens, Blo t 5 and Der p 5, remains controversial. OBJECTIVE: This study was undertaken to assess the levels of the IgE cross-reactivity between Blo t 5 and Der p 5 by using sera from a large cohort of asthmatic children in subtropical and tropical countries. METHODS: Purified recombinant Blo t 5 and Der p 5 were produced in Pichia pastoris and tested against sera from 195 asthmatic children. The IgE cross-reactivity was examined by direct, inhibitory and competitive human IgE enzyme-linked immunosorbent assay as well as skin prick tests. RESULTS: The Blo t 5 IgE responses were 91.8% (134 of 146) and 73.5% (36 of 49) for Taiwanese and Malaysian sera, respectively. The Blo t 5 specific IgE titers were significantly higher than those of Der p 5 (P <.02). The correlation of IgE reactivity between Blo t 5 and Der p 5 was low, and only limited cross-reactivity was observed. This was further confirmed by the dose-response inhibition studies. Skin prick tests performed on asthmatic children in Thailand also showed differential IgE response to Blo t 5 and Der p 5. CONCLUSION: By using a large panel of asthmatic sera and a combination of in vitro and in vivo assays, the major allergen of B tropicalis in tropical and subtropical regions, Blo t 5, exhibits low levels of IgE cross-reactivity with homologous Der p 5. These findings suggest that highly specific clinical reagents are necessary for precise diagnosis and immunotherapeutic treatment of sensitization to group 5 mite allergens.     

Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties

Alginate gels have been used in both drug delivery and cell encapsulation applications in the bead form usually produced by dripping alginate solution into a CaCl2 bath. The major disadvantages to these systems are that the gelation rate is hard to control; the resulting structure is not uniform; and mechanically strong and complex-shaped 3-D structures are difficult to achieve. In this work controlled gelation rate was achieved with CaCO3-GDL and CaSO4-CaCO3-GDL systems, and homogeneous alginate gels were formulated as scaffolds with defined dimensions for tissue engineering applications. Gelation rate increased with increasing total calcium content, increasing proportion of CaSO4, increasing temperature and decreasing alginate concentration. Mechanical properties of the alginate gels were controlled by the compositional variables. Slower gelation systems generate more uniform and mechanically stronger gels than faster gelation systems. The compressive modulus and strength increased with alginate concentration, total calcium content, molecular weight and guluronic acid (G) content of the alginate. MC3T3-E1 osteoblastic cells were uniformly incorporated in the alginate gels and cultured in vitro. These results demonstrated how alginate gel and gel/cell systems could be formulated with controlled structure, gelation rate, and mechanical properties for tissue engineering and other biomedical applications.     

Influence of active dendritic currents on input-output processing in spinal motoneurons in vivo

The extensive dendritic tree of the adult spinal motoneuron generates a powerful persistent inward current (PIC). We investigated how this dendritic PIC influenced conversion of synaptic input to rhythmic firing. A linearly increasing, predominantly excitatory synaptic input was generated in triceps ankle extensor motoneurons by slow stretch (duration: 2-10 s) of the Achilles tendon in the decerebrate cat preparation. The firing pattern evoked by stretch was measured by injecting a steady current to depolarize the cell to threshold for firing. The effective synaptic current (I(N), the net synaptic current reaching the soma of the cell) evoked by stretch was measured during voltage clamp. Hyperpolarized holding potentials were used to minimize the activation of the dendritic PIC and thus estimate stretch-evoked I(N) for a passive dendritic tree (I(N,PASS)). Depolarized holding potentials that approximated the average membrane potential during rhythmic firing allowed strong activation of the dendritic PIC and thus resulted in marked enhancement of the total stretch-evoked I(N) (I(N,TOT)). The net effect of the dendritic PIC on the generation of rhythmic firing was assessed by plotting stretch-evoked firing (strong PIC activation) versus stretch-evoked I(N,PASS) (minimal PIC activation). The gain of this input-output function for the neuron (I-O(N)) was found to be ~2.7 times as high as for the standard injected frequency current (F-I) function in low-input conductance neurons. However, about halfway through the stretch, firing rate tended to become constant, resulting in a sharp saturation in I-O(N) that was not present in F-I. In addition, the gain of I-O(N) decreased sharply with increasing input conductance, resulting in much lower stretch-evoked firing rates in high-input conductance cells. All three of these phenomena (high initial gain, saturation, and differences in low- and high-input conductance cells) were also readily apparent in the differences between stretch-evoked I(N,TOT) and I(N, PASS) and thus could be accounted for by the activation of the dendritic PIC. As a result, stretch-evoked I(N,TOT) and F-I provided an accurate prediction of the overall change in stretch-evoked firing. However, in about half of the low-input conductance cells, the rate of rise of firing in response to stretch was not smoothly graded but instead consisted of a rapid surge. Stretch-evoked I(N,TOT) was always smoothly graded. This suggests that although stretch-evoked I(N,TOT) can be used to predict the overall change in firing, prediction of the dynamics of firing may be less accurate.     

Serological response to Chlamydia pneumoniae infection.

The human serological response was analyzed by using sera from patients who were serologically positive but isolation negative for Chlamydia pneumoniae and from patients with proven C. pneumoniae infection based on serology and isolation. To assess whether seroreactivity to C. pneumoniae proteins had potential diagnostic value, the cross-reactivities of these sera to other Chlamydia species and of sera from patients infected with C. trachomatis and C. psittaci to C. pneumoniae proteins were determined. In all serum samples from patients with proven C. pneumoniae infections, reactivities were seen with 98-, 68-, 60-, 39.5-, and 30-kilodalton proteins. Similar patterns were seen in sera from patients who were serologically positive and isolation negative. The onset of seropositivity for C. pneumoniae was accompanied by reactivities against presumably shared chlamydial antigens and a C. pneumoniae-specific 98-kilodalton protein.     

Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium-aggravated myotonia, long QT–3 syndrome, and neuropathic pain. This drug elicits tonic block of Na⁺ channels when cells are stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation-deficient mutant Na⁺ channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a β1 subunit. The 50% inhibitory concentrations (IC₅₀) for the inactivated-state block and the resting-state block of wild-type Na⁺ channels by mexiletine were measured as 67.8 ± 7.0 μ m and 431.2 ± 9.4 μ m , respectively ( n = 5). In contrast, the IC₅₀ for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 μ m ( n = 5), which was within the therapeutic plasma concentration range (2.8–11 μ m ). Estimated on- and off-rates for the open-state block by mexiletine at +30 mV were 10.4 μ m ⁻¹ s⁻¹ and 54.4 s⁻¹, respectively. Use-dependent block by mexiletine was greater in inactivation-deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC₅₀ values for the block of persistent late hNav1.4 currents in chloramine-T-pretreated cells by mexiletine was 7.5 ± 0.8 μ m ( n = 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open-channel block of persistent late Na⁺ currents, which may arise during various pathological conditions.