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The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells produced less IL-12p70 and IFN-α (and consequently induced less IFN-γ), moderately less TNF-α, but as much or even more IL-1β, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs the adult white-blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell-mediated in vivo control of HIV-1. Primary HIV-1-specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or "vertical" immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.

Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20–40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.

In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants., Author Summary An important role for the polyfunctional T-cell fraction of anti-HIV CD8 responses during chronic HIV infection has previously been suggested. This study characterized the role of polyfunctional T-cells directed against the transmitted/founder virus in the selection of viral escape mutants during acute HIV-1 infection within a unique cohort of individuals recruited within 3 weeks from the onset of symptoms at the time when the virus load was still declining. For the first time, the sequences of the transmitted/founder virus isolated from each patient were used. Interestingly, polyfunctionality was not found to be a pre-requisite for selection of escape mutations. A novel significant correlation is found between the order of appearance of escape mutations in different epitope sequences and both the magnitude of the CD8+ T-cell responses and the degree of entropy of the individual epitopes. A high proportion of the T-cells participating in the total response produced MIP-1β, suggesting that mechanisms not limited to the killing of infected cells might play a relevant role in early infection. This highlights the importance of measuring the quality of the CD8+ lymphocyte response and the sequence of the transmitted virus isolates to better understand the mechanisms of control of HIV replication during acute infection.

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1 + ) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1 + plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.

Contradictory findings from studies on pretreatment malalignment as a risk factor for relapse of maxillary incisor alignment may be due to inappropriate sample selection and measurement technique. In an attempt to clarify the issue, 745 sets of study models made before (T1) and after (T2) orthodontic treatment and at long-term out of retention (T3) were screened. On the basis of the configuration of the maxillary anterior teeth on the T3 study models, three groups were established: one with significant spacing (group 1, n = 30); one with significant irregularity (group 2, n = 49); and one with perfect alignment (group 3, n = 28). The occlusal surfaces of the 321 maxillary study models at T1, T2, and T3 were photocopied and the tooth anatomic contact points digitized. An algorithm was used to fit the dental arch to the digitized points. Amount of incisor rotation and anatomic contact point displacement of the maxillary anterior teeth relative to the dental arch were computer generated. Interdental spaces in the maxillary anterior segment, as well as overjet and overbite, were measured manually. Nonstructural data were collected from the charts. Logistic regression analyses revealed that irregularity was associated with greater anatomic contact displacement and with greater incisor rotation both at T1 and T2 (P0.01). Similar analyses also revealed that spacing was associated with greater interdental spaces at T1 and T2 (P0.01). Correlation analyses revealed that the pattern of pretreatment rotational displacement has a strong tendency to repeat itself after retention (P0.001), as opposed to the pattern of contact point displacement and interdental spacing.

The purpose of this investigation was to determine whether the presence of disease-associated bacteria in health-associated plaque correlated with susceptibility to periodontitis over time. Sites of periodontal health were identified in 65 adults. Six months later (recall 1), plaque was collected from sites that remained in periodontal health, and specific bacteria were detected using monoclonal antibodies in a microscopic assay. The spirochete morphogroup was identified by phase contrast microscopy. The relationship between detection at recall 1 and development of periodontitis over two successive 6-month intervals (recalls 2 and 3) was evaluated by means of logistic regression using generalized estimating equations (GEE), from which odds ratios (OR) were estimated and tested for significance. Significant relationships were defined as those having ORs with P < 0.05. Ninety-three of 1,032 sites developed signs of early periodontitis over the 12-month interval between recall 1 and recall 3. The spirochete morphogroup (OR = 3.13, P < 0.001) and pathogen-related oral spirochetes (PROS) (OR = 3.68, P < 0.001) were significantly associated with healthy sites that developed periodontitis. The association of Treponema socranskii was not significant (OR = 3.62, P = 0.0918). Odds ratios for Campylobacter rectus, Eikenella corrodens, and Porphyromonas gingivalis were less than 2.0 and not significant. Treponema denticola was not detected in health-associated plaque from stable health sites and was detected in only three sites that progressed to periodontitis. These findings indicate that the presence of PROS and some unidentified spirochetes in health-associated plaque is associated with increased susceptibility to periodontitis.

Intra-abdominal adhesions have been shown to result from the impairment of peritoneal fibrinolysis by inhibitors present in ischemic tissue. A reproducible model for the formation of intra-abdominal adhesions was utilized for the evaluation of the effectiveness of intraperitoneal applications of recombinant tissue plasminogen activator (rtPA) in adhesion prevention. Concentrations of rtPA required to overcome the inhibition of fibrinolysis in this model were estimated by titration of that amount of rtPA required to lyse blood clot in the presence of a measured amount of ischemic tissue. Adhesions were graded, and the hydroxyproline content of the abdominal wounds was analyzed. The effect of intraperitoneal administration of rtPA on adhesion formation was strongly dose related. Levels of rtPA of 0.01 mg/mL showed no effect (p0.75) on adhesion formation, whereas levels of 0.1 mg/mL either prevented or significantly modified the formation of intra-abdominal adhesions (p0.05). Concomitantly, wound hydroxyproline content was significantly reduced (p = 0.004). Prior investigations have shown a strong correlation between wound bursting strength and hydroxyproline content. The results of this study indicated that the levels of rtPA required to alter or prevent intra-abdominal adhesion formation also produce a significant impairment of the early phase of wound healing as measured by the wound content of hydroxyproline.

Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life. A more complete understanding of the ontogeny of the immune system over the first years of life is thus urgently needed. Here, we applied the most comprehensive analysis focused on the innate immune response following TLR stimulation over the first 2 years of life in the largest such longitudinal cohort studied to-date (35 subjects). We found that innate TLR responses (i) known to support Th17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood; (ii) potentially supporting antiviral defense (IFN-α) reached adult level function by 1 year of age; (iii) known to support Th1 type immunity (IL-12p70, IFN-γ) slowly rose from a low at birth but remained far below adult responses even at 2 years of age; (iv) inducing IL-10 production steadily declined from a high around birth to adult levels by 1 or 2 years of age, and; (v) leading to production of TNF-α or IL-1β varied by stimuli. Our data contradict the notion of a linear progression from an 'immature' neonatal to a 'mature' adult pattern, but instead indicate the existence of qualitative and quantitative age-specific changes in innate immune reactivity in response to TLR stimulation.

Statement of Problem. Little is known about the importance of published fabrication standards in determining the outcome of treatment with mandibular distal extension removable partial dentures in patients of community practices. Purpose. This study describes mandibular partial dentures worn by patients from King County, Wash., and examines the validity of standards of design and fabrication by relating the standards to measures of clinical acceptability and patient satisfaction. Material and Methods. Eighty-two people treated in private dental practices who responded to a mail survey about satisfaction with a partial denture were examined. Eight standards of fabrication, overall clinical acceptability of the prosthesis, and tissue health were evaluated clinically. Patient satisfaction was assessed by questionnaire. Associations between variables were assessed by contingency tables and odds ratios. Results. Half the prostheses met 4 or fewer of the 8 standards, and these were responsible for nearly all the displacement of tissue by the framework. Forty-three percent of the dentures (35/82) were rated clinically acceptable. Of the remaining dentures, 38 could be made acceptable by modifications. The remaining 9 dentures needed replacement. Sixty-three percent of the patients examined were satisfied with the dentures. There was a relationship between tissue health and the fabrication standards related to rest form, base extension, stress distribution, and framework fit. This study found no relation between tissue health and other design or fabrication features. None of the standards were found to be related to patient satisfaction. Conclusion. This study found partial support for the validity of design/fabrication standards for removable partial dentures. The most important standards are rest form, base extension, and stress distribution. The standards appear to be unrelated to patient satisfaction. (J Prosthet Dent 2000;83:521-7.)

Background One of the greatest challenges to develop an efficacious HIV vaccine is the enormous diversity of HIV-1. To tackle this problem, T cells based vaccine approaches have come up with two main camps: the mosaic immunogen camp: increasing the breadth of vaccine-induced responses, and the conserved immunogen camp: targeting vaccineinduced T-cell responses only to highly conserved viral regions. While both approaches are theoretically sound, there is no current data suggesting that either approach will be successful in inducing T cells with superior antiviral efficacy. Here we analyzed T cell responses elicited during early HIV-1 infection, to address the question whether CS of targeted epitopes and breadth of T cell responses play an important role in viral control. Methods Using IFN-g ELISpot, we comprehensively mapped T cell epitope specificities recognized by 24 ART-naive individuals during early infection. We identified CS of targeted epitopes, where the CS is defined as the proportion of random HIV-1 group M amino acid sequences in the LANL database that include the epitope. We used a prediction model to impute the viral load (VL) set-point using the first available VL as a predictor for subjects lacking VL set-point. We further evaluated the association between the CS of the targeted epitopes and breadth of T cell responses to the individuals’ VL set-point.