Acylation of the lipooligosaccharide of Haemophilus influenzae and colonization: an htrB mutation diminishes the colonization of human airway epithelial cells

Haemophilus influenzae is a commensal and opportunistic pathogen of the human airways. A number of surface molecules contribute to colonization of the airways by H. influenzae, such as adhesins, including structures found in the lipooligosaccharide (LOS). A human bronchiolar xenograft model was employed to investigate the host-bacterial interactions involved in the colonization of the airway by H. influenzae. Differential display was used to identify H. influenzae mRNA that reflect genes which were preferentially expressed in the xenograft compared to growth. Eleven mRNA fragments had consistent increased expression when the bacteria grew in xenografts. On sequencing these fragments, eight open reading frames were identified. Three of these had no match in the NCBI or the TIGR database, while an additional three were homologous to genes involved in heme or iron acquisition and utilization: two of the mRNAs encoded proteins homologous to enzymes involved in LOS biosynthesis: a heptosyl transferase (rfaF) involved in the synthesis of the LOS core and a ketodeoxyoctonate phosphate-dependent acyltransferase (htrB) that performs one of the late acylation reactions in lipid A synthesis. Inoculation of human bronchiolar xenografts revealed a significant reduction in colonization capacity by htrB mutants. In vitro, htrB mutants elicited lesser degrees of cytoskeletal rearrangement and less stimulation of host cell signaling with 16HBE14o(-) cells and decreased intracellular survival. These results implicate acylation of H. influenzae lipid A as playing a key role in the organisms' colonization of the normal airway.     

Baroreflex function in lifetime-captopril-treated spontaneously hypertensive rats

The effects of lifetime oral captopril treatment on baroreflex control of heart rate and lumbar sympathetic nerve activity were measured in 19-21-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The sensitivity of baroreflex control of heart rate and lumbar sympathetic nerve activity were determined by the slopes of the relation between the change in mean arterial pressure (MAP) (mm Hg) versus the change in pulse interval (msec/beat) and the change in MAP versus the percent change in nerve activity, respectively. Untreated SHR had significantly higher MAP than WKY (157 +/- 3 vs. 115 +/- 3 mm Hg, p less than 0.001) and exhibited a decreased baroreflex control of heart rate. Lifetime treatment with captopril prevented the development of hypertension in SHR (MAP = 110 +/- 5 mm Hg) and increased the sensitivity of baroreflex function. The gains of the baroreflex control of heart rate for captopril-treated SHR and control SHR when MAP was raised or lowered by phenylephrine or nitroprusside were 2.38 +/- 0.49 vs. 1.10 +/- 0.33 msec/mm Hg (p less than 0.05) and 0.74 +/- 0.20 vs. 0.54 +/- 0.09 (NS) msec/mm Hg, respectively. The sensitivity of the baroreflex control of lumbar sympathetic nerve activity was greater in captopril-treated SHR than in control SHR when MAP was increased or decreased (-1.03 +/- 0.26 vs. -0.38 +/- 0.11, p less than 0.05; -0.84 +/- 0.2 vs. -0.04 +/- 0.58 (NS) mm Hg-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and direct sequencing from lambda cDNA libraries using the polymerase chain reaction: suppressin and the vasopressin receptor as models.

A strategy using the polymerase chain reaction (PCR) to screen a lambda gt11 pituitary cDNA library for cDNAs encoding suppressin, a putative anti-proliferative protein, and a putative vasopressin receptor is described. The use of this technique will facilitate the demonstration of e.g. the presence of "neuropeptide receptors" on cells of the lymphoid system, confirming the concept of "shared ligands and receptors" by the neuroendocrine and the immune system. Neither of the genes encoding the proteins of the present study have previously been cloned. The PCR-screening procedure requires sequence information from the gene of interest which permits the generation of complementary primers. These primers are then used in combination with lambda phage primers complementary to regions flanking the cloning site in a PCR to amplify cDNAs derived from the gene of interest. This novel screening procedure yields cDNA related to the gene of interest, including the largest clone present in the library. To confirm the utility of this technique for cDNA libraries, the library was also screened using traditional cDNA hybridization techniques. The largest clone obtained by screening the cDNA library with PCR was the same as that obtained by the conventional technique. Thus, the results of these studies show that the PCR method can be used instead of more conventional means to screen cDNA libraries. Lastly, we describe a protocol for directly sequencing PCR-amplified DNA using the same primers that are used for amplification. The combined use of these two strategies permits cloning and sequencing of cDNAs from lambda cDNA libraries in a fraction of the time required using traditional screening techniques, but with identical results.     

Interleukin-10 promoter polymorphisms in rheumatoid arthritis and Felty's syndrome

OBJECTIVE: To examine whether promoter polymorphisms associated with variation in interleukin-10 (IL-10) production are relevant to the development of rheumatoid arthritis (RA) or Felty's syndrome (FS). METHODS: DNA was obtained from 44 FS patients, 117 RA patients and 295 controls. The promoter region between -533 and - 1120 was amplified by polymerase chain reaction, and polymorphisms detected by restriction enzyme digest or sequence-specific oligonucleotide probing. RESULTS: We found no significant difference in allele or haplotype frequencies between the groups. CONCLUSION: There is no association between FS or RA and these recently identified IL-10 promoter polymorphisms. Other genetic or environmental factors could explain the alterations in IL-10 levels seen in these conditions.