Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation

Cytokine-producing cells in gut-associated lymphoid tissues of rhesus macaques with chronic enterocolitis were studied. The confocal microscopy technique that we developed enables simultaneous in situ visualization of multiple extra- and/or intracellular antigens at a resolution higher than that allowed by light or epifluorescence microscopy. The presence of interleukin-6 (IL-6)-, tumor necrosis factor alpha (TNF-alpha)-, and IL-1-alpha-producing cells was focally intense in the colon lamina propria of the affected animals. The IL-1-alpha-producing cells were T lymphocytes (CD3+), while the TNF-alpha-producing cells were both macrophages (CD68+/HAM56+/LN5+) and T lymphocytes (CD3+). The IL-6-producing cells within the colon consisted of T lymphocytes and macrophages. The amount of IL-6-producing cells seen in macaques with enterocolitis was significantly higher (P<0.001) than that seen in the healthy control animal, while TNF-alpha- and IL-1-alpha-producing cells were seen only in macaques with enterocolitis. Most of the T lymphocytes that produced cytokines were detected in the lamina propria, while the macrophages were most prominent in highly inflamed regions of the lamina propria. Taken together, our findings indicate that there might be immunological similarity between chronic enterocolitis of rhesus macaques and humans, suggesting the potential use of the nonhuman primate model for the validation of novel therapies.     

Human mtDNA hypervariable regions, HVR I and II, hint at deep common maternal founder and subsequent maternal gene flow in Indian population groups

We have analysed the hypervariable regions (HVR I and II) of human mitochondrial DNA (mtDNA) in individuals from Uttar Pradesh (UP), Bihar (BI) and Punjab (PUNJ), belonging to the Indo-European linguistic group, and from South India (SI), that have their linguistic roots in Dravidian language. Our analysis revealed the presence of known and novel mutations in both hypervariable regions in the studied population groups. Median joining network analyses based on mtDNA showed extensive overlap in mtDNA lineages despite the extensive cultural and linguistic diversity. MDS plot analysis based on Fst distances suggested increased maternal genetic proximity for the studied population groups compared with other world populations. Mismatch distribution curves, respective neighbour joining trees and other statistical analyses showed that there were significant expansions. The study revealed an ancient common ancestry for the studied population groups, most probably through common founder female lineage(s), and also indicated that human migrations occurred (maybe across and within the Indian subcontinent) even after the initial phase of female migration to India.     

Glycolipids of Mycobacterium tuberculosis Strain H37Rv Are Potential Serological Markers for Diagnosis of Active Tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

Personal relationships with an intelligent interactive telephone health behavior advisor system: a multimethod study using surveys and ethnographic interviews

The burgeoning of consumer health informatics and virtual health care can help people improve their health. However, little is known about individuals' reactions to such systems. We conducted an evaluation of the telephone-linked care (TLC) system, a computer-based telecommunications system, that functions as an at home monitor, educator, and counselor for patients with chronic health conditions. Our multimethod assessment of individuals' reactions to using TLC included both quantitative and qualitative methods. Ethnographic in-depth open-ended interviews indicated more subtle and surprising reactions to TLC than the overall positive responses from surveys: individuals formed personal relationships with this technology. This relationship formation suggests that TLC designers may have been successful in their attempts to emulate a conversation with a human being. Our study adds to evidence that technology can serve as a projective device for peoples' values and psychological issues. Both designers and users project values and goals onto computer-based technologies and take on different identities through it. Different groups of users, therefore, may see the same technology differently. People also form relationships with technologies, as they did with TLC. These findings, as well as implications for system design and health outcomes, need to be explored in additional studies.     

The murine NF2 homologue encodes a highly conserved merlin protein with alternative forms

The recently isolated gene for neuroflbromatosls type 2 (NF2)encodes a 595 amlno acid protein, named merlin, which Is relatedto the cytoskeleton-assoclated proteins moesln, ezrin and radlxin.To Identify evolutionarily conserved regions and to providesequence Information necessary for the establishment of a mousemodel for NF2, we have determined the cDNA sequence of the mouseNF2 tumor suppressor gene, and mapped It In the mouse genome.Mouse merlin is a 596 amino acid protein, 98% identical to humanmerlin, but one amlno acid longer due to the Insertion of aproline residue near the C-terminus. Of the nine amlno aciddifferences between mouse and humans, seven occur in the C-termlnal20% of the protein, far from the protein 4. 1 domain that definesthis family. Two of the NF2 cDNA clones reveal evidence of alternativesplicing events that alter the predicted merlin product, oneremoving a 45 amlno acid segment from the middle section ofthe protein and the other changing the C-terminus. The existenceof several different forms of merlin potentially with differentprimary roles will complicate the Identification of the precisefunction that must be disrupted to cause the NF2-assoclatedtumors. The mouse NF2 homologue maps to Chr 11, in a regionhomologous to human Chr 22, but devoid of any mouse mutationswhich could be models of the human disorder.     

Roles of specific amino acids in the N terminus of Pseudomonas aeruginosa flagellin and of flagellin glycosylation in the innate immune response

The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/mug) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flagellin.     

A human recessive neurosensory nonsyndromic hearing impairment locus is a potential homologue of the murine deafness (dn) locus

A locus for recessive neurosensory nonsyndromic hearing impairmentmaps to chromosome 9q13–q21 in two regionally separateconsanguineous families from India. Each family demonstratesa LOD score greater than 4.5 to this region. D9S15, tightlylinked to the Friedreich's ataxia locus, a region that has beendefined with over 1 Mb of YAC contig information and severalexpressed sequences, is one of the flanking markers. In mice,the deafness (dn) locus maps to mouse chromosome 19 and flankingloci are syntenic to human chromosome 9q11–q21. The dnmouse is a potential model for the hearing impairment foundin both these families.     

Human papillomavirus DNA in urine samples of women with or without cervical cancer and their male partners compared with simultaneously collected cervical/penile smear or biopsy specimens.

Infection of specific types of high-risk human papillomaviruses (HPVs) causes cervical cancer in women. Conventional test for genital HPV infection requires collection of scraped cervical cells or biopsy specimens, which involves invasive procedures. Utility of non-invasive urine sampling for detection of HPV in women and their male sexual partners is controversial. The validation of this urine-based HPV DNA test is of immense value not only in screening large population and children but also for HPV vaccine monitoring in adolescents. We examined the frequency of high risk HPV types 16 and 18 in simultaneously collected urine samples and cervical scrapes or biopsy specimens from women with cervical cancer and their single lifetime male sexual partners in order to validate the utility of urine sampling as a reliable non-invasive method for detection of genital HPV infection. Thirty women with invasive cervical cancer and their husbands along with 30 age-matched normal healthy women including their husbands were recruited for the study. Cervical biopsies/scrapes from women subjects and penile scrapes from their husbands and urine samples from all of them were collected before taking biopsy or scrapes. HPV-L1 consensus primer as well as high-risk HPV (HPV 16 and 18) type-specific oligo-primers were used for PCR detection of HPV DNA. The total frequency of HPV in women with cervical cancer was found to be 83% (25/30) while it was only 67% (20/30) in their male partners but there was virtually no difference in results between urine and scrape or tissue biopsy either in women or their male partners. Although healthy women and their husbands showed similar frequency of HPV infection both in urine and scrape samples, there was a significant difference (p=0.05) in the prevalence of high risk HPV type 16 in women with cervical cancer (70%) and their male partners (30%). Similar was the trend between control women and their male partners. The results also showed a very high prevalence of HPV type 16 among Indian women with cervical cancer while its frequency was significantly low in their single lifetime male partners. The case by case matching of HPV positivity and negativity between urine and cervical/penile scrapes or biopsies obtained from women and their male partners demonstrated that the non-invasive urine sampling can be reliably used for screening genital HPV infection in both men and women.     

Microbial flora in orodental infections

The present study was carried out to compare the normal aerobic and anaerobic bacterial oral flora with flora from deep seated dental caries, gingivitis and adult periodontitis. All the samples belonging to both the control and study groups yielded microbes. Aerobe / Anaerobe ratio was high in normal flora (1.48) as compared to dental caries (0.9), gingivitis (0.72) and periodontitis (0.56). Ninety seven percent of orodental infections were polymicrobial and three or more microbes were found in 84% cases of study group as compared to 28% in controls. Streptococcus mutans and anaerobic lactobacilli were common in dental caries, Actinomyces and Peptostreptococcus spp. in gingivitis, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in periodontitis.