Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Antibiotic use and hygiene interact to influence the distribution of antimicrobial-resistant bacteria in low-income communities in Guatemala.

To examine the effects of poor sanitation and hygiene on the prevalence of antimicrobial-resistant bacteria, we surveyed households in two rural and two urban communities in Guatemala (N = 196 randomly selected households). One adult (≥ 18-years old) and, when available, one child (≤ 5 years-old) provided a stool sample. Up to 48 presumptive Escherichia coli isolates were collected from each stool sample (n = 21,256 total) and were subjected to breakpoint assays for ten antibiotics. Mixed-effects logistic models were used to identify potential factors influencing the likelihood of harboring antibiotic-resistant bacteria. For nine out of ten antibiotics, the odds of detecting resistant bacteria decreased by ~ 32% (odds ratios, OR 0.53-0.8, P < 0.001) for every unit of improvement of a hygiene scale. Hygiene differences between households had a greater impact on prevalence compared to antibiotic use differences. The likelihood of detecting resistant isolates was lower for five antibiotics among households that boiled raw milk before consumption (OR 0.31-0.69), and higher for nine antibiotics in urban households (OR > 1.89-9.6). Poor hygiene conditions likely obscure effects of individual antibiotic use, presumably due to enhanced microbial transmission. Consequently, efforts to improve antibiotic stewardship should be coupled with improving hygiene conditions.

Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4(+) T cell epitopes with other tree nuts.

BACKGROUND: Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T cell epitopes and T cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. OBJECTIVES: In this study, we characterized cashew-specific T cell responses in cashew-allergic subjects and examined cross-reactivity of these cashew-specific cells towards other tree nut allergens. METHODS: CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T cells were determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. RESULTS: CD4(+) T cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. CONCLUSIONS: Phylogenetically diverse tree nut allergens can activate cashew-reactive T cells and elicit a TH 2-type response at an epitope-specific level. CLINICAL RELEVANCE: Lack of cross-reactivity between walnut and cashew suggests that cashew peptide immunotherapy approach may not be most effective for walnut.

Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

BACKGROUND: Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity among grass-pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. OBJECTIVES: We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4(+) T cell level among DR04:01 restricted Pooideae grass-pollen T-cell epitopes. METHODS: After in vitro culture of blood mono-nucleated cells from grass-pollen-allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labelled DR04:01/Phleum pratense tetramers and PE-labelled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity among allergen-specific DR04:01-restricted T-cells in six subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass-pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. RESULTS: T-cells with various degrees of cross-reactive profiles could be detected. Poa p 1 97-116 , Lol p 1 221-240 , Lol p 5a 199-218 , and Poa p 5a 199-218 were identified as minimally cross-reactive T-cell epitopes that do not show cross-reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally cross-reactive T-cell epitopes are present in Grass-pollen-allergic subjects. CONCLUSIONS: Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross-reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells suggest that a multiple allergen system should be considered for immunotherapy instead of a mono-allergen system.

Subgingival microbiota of indigenous Indians of Central America.

OBJECTIVE: To define the subgingival microbial profiles of adult subjects from a previously identified rural community of indigenous Indians in Guatemala, Central America. MATERIALS AND METHODS: A full-mouth periodontal examination was performed in 114 adult subjects from 45 families. Plaque samples were collected from both deep and shallow periodontal pockets and checkerboard DNA-DNA hybridization was employed to identify 17 species previously associated with periodontitis or health. RESULTS: Plaque deposits and gingivitis were universal and widespread, and periodontal pocketing > or =5 mm was highly prevalent (84% of subjects). Streptococcus sanguis, Actinomyces naeslundii genospecies 2 and Fusobacterium nucleatum were significantly more prevalent in shallow sites. At the subject level, Actinomyces naeslundii and Peptostreptococcus micros were significantly more prevalent in periodontally-healthy subjects. Actinobacillus actinomycetemcomitans was not detected in any sample. CONCLUSION: There was no association between periodontal disease status and presence of suspected periodontal pathogens. These latter results conflict somewhat with those from treated populations. However, in this population where extensive plaque deposits and gingivitis are universal, the presence of putative pathogens may be more reflective of the local environment.

Oral health status of an indigenous adult population of Central America.

OBJECTIVE: To determine the caries experience and periodontal disease status in adults of an indigenous rural community of Guatemala, and assess the suitability for longitudinal investigations. BASIC RESEARCH DESIGN: This investigation comprised an initial screen (Study I) and a more detailed periodontal examination (Study II). In Study I, caries and gingivitis levels were determined. In Study II, pocket probing depths (PPDs) and clinical attachment levels (CALs) were recorded on all teeth excluding third molars. CLINICAL SETTING: Tzununa, Guatemala, Central America. PARTICIPANTS: Studies I and II were conducted in 120 adults 3 18 years and 54 adults 3 25 years respectively. RESULTS: In both Studies I and II, tooth retention was high with a mean tooth count of 28.2 and 27.2 respectively. Extensive soft deposits and both supra- and subgingival calculus were almost universal, although gingivitis was less than expected (Study I: Mean percentage of sites bleeding on probing = 27.6). In Study I, the mean number of carious teeth was 8.6 and there was no statistically significant correlation with age. In Study II, PPD 3 5mm and CAL 3 6mm were highly prevalent (100% and 56% of subjects respectively), although widespread and severe disease was not evident. CONCLUSIONS: Despite the high caries level and the evidence of periodontal destruction in the majority of subjects, all study subjects had a functional dentition suggesting that emergency treatment remains the current priority. Longitudinal studies in such untreated populations would provide increased understanding of the role of environmental factors in disease etiology. The study also highlighted some methodological issues pertinent to conducting studies in remote communities.

Periodontal disease status of an indigenous population of Guatemala, Central America.

OBJECTIVES: This study was designed to determine the periodontal disease status of an indigenous Indian community of rural Central America (San Juan La Laguna, Guatemala), for comparison with results of similar studies in other populations, and with a view to performing future studies to address familial clustering of adult periodontitis. METHODS & RESULTS: An initial screen of 239 subjects aged 12-75 years from extended families suggested a high disease prevalence according to full-mouth pocket probing depths (PPDs), with more than 75% of subjects with one or more pockets of PPD > or =5 mm. A more detailed study was performed in 125 unrelated subjects > or =18 years, recording full-mouth PPDs and clinical attachment levels (CALs). The high prevalence of pocketing was confirmed and 90% of adults > or =35 years had at least one site with CAL > or =6 mm. However, extensive disease was restricted to a small minority, with only 10% of adults > or =35 years having 20% or more sites with CAL > or =6 mm. CONCLUSION: The study results highlight the importance of performing a detailed examination and appropriate analysis. In both studies, tooth retention was high (mean number of teeth recorded was 26.4 and 28.0 respectively), smoking unusual, and families large and localised to the village. This community thus affords several advantages over populations in developed countries when considering familial studies of adult periodontitis.

Helicobacter pylori infection in indigenous families of Central America: serostatus and oral and fingernail carriage.

Helicobacter pylori infection remains one of the most common in humans, but the route of transmission of the bacterium is still uncertain. This study was designed to elucidate possible sources of infection in an isolated, rural population in Guatemala. A total of 242 subjects in family units participated in the study. A medical history, including a history of dyspepsia, was taken by a physician and immunoglobulin G antibodies to H. pylori were detected with the QuickVue (Quidel, San Diego, Calif.) onsite serology test. Overall, 58% of subjects were seropositive, with a positive relationship between mother and child (P = 0.02) and a positive correlation between the serostatuses of siblings (intraclass correlation coefficient = 0.63). There was no association between serostatus and gastric symptoms. Oral H. pylori was detected from periodontal pockets of various depths and the dorsum of the tongue by nested PCR. Eighty-seven percent of subjects had at least one oral site positive for H. pylori, with the majority of subjects having multiple positive sites. There was no association between periodontal pocket depth and the detection of H. pylori. Nested PCR was also used to detect H. pylori from beneath the nail of the index finger of each subject's dominant hand. Overall, 58% of subjects had a positive fingernail result, with a significant positive relationship between fingernail and tongue positivity (P = 0.002). In conclusion, the results of this study suggest that oral carriage of H. pylori may play a role in the transmission of infection and that the hand may be instrumental in transmission.