Some lessons from the systematic production and structural analysis of soluble (alpha)(beta) T-cell receptors.

T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, (alpha)(beta)-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes.

Aggressive strategic planning for oral health in Kuwait: a decade of post-war successes.

Strategic planning and implementation of oral health care and disease prevention programmes after the 1990/91 Gulf war are discussed. The key concept was to develop access to care and disease prevention for all Kuwaiti children in government kindergarten/primary schools and to eliminate emphasis on extractions and restorations. Resources were restored to pre-war levels and then increased. Prevention programmes for 150 000 children were established. Prevention funds increased from 7% to 20% of the oral health budget. Prevention-based dentists increased from 9.7% to 28.0% of staff. Rising caries trends were stabilized or reduced by up to 36.8%. Percentage of caries-free primary dentition in children increased up to 37.6%, permanent dentition up to 27.0%. A dentistry school was established.

Aggressive strategic planning for oral health in Kuwait: a decade of post-war successes

Strategic planning and implementation of oral health care and disease prevention programmes after the 1990/91 Gulf war are discussed. The key concept was to develop access to care and disease prevention for all Kuwaiti children in government kindergarten/primary schools and to eliminate emphasis on extractions and restorations. Resources were restored to pre-war levels and then increased. Prevention programmes for 150 000 children were established. Prevention funds increased from 7% to 20% of the oral health budget. Prevention-based dentists increased from 9.7% to 28.0% of staff. Rising caries trends were stabilized or reduced by up to 36.8%. Percentage of caries-free primary dentition in children increased up to 37.6%, permanent dentition up to 27.0%. A dentistry school was established

Comparison between HIV- and CMV-specific T cell responses in long-term HIV infected donors.

The mechanisms underlying non-progression in HIV-1 infection are not well understood; however, this state has been associated previously with strong HIV-1-specific CD8+ T cell responses and the preservation of proliferative CD4+ T cell responses to HIV-1 antigens. Using a combination of interferon-gamma (IFN-gamma) ELISpot assays and tetramer staining, the HIV-1-specific CD8+ T cell populations were quantified and characterized in untreated long-term HIV-1-infected non-progressors and individuals with slowly progressive disease, both in relation to CD4+ T cell responses, and in comparison with responses to cytomegalovirus (CMV) antigens. High levels of CD8+ T cell responses specific for HIV-1 or CMV were observed, but neither their frequency nor their phenotype seemed to differ between the two patient groups. Moreover, while CMV-specific CD4+ T cell responses were preserved in these donors, IFN-gamma release by HIV-1-specific CD4+ T cells was generally low. These data raise questions with regard to the role played by CD8+ T cells in the establishment and maintenance of long-term non-progression.

Functional differences between influenza A-specific cytotoxic T lymphocyte clones expressing dominant and subdominant TCR.

We have shown that the dominance of CD8+ T cells expressing TCR Vbeta17 in the adult HLA-A*0201-restricted influenza A/M1(58-66)-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vbeta17+ cells, subdominant M1(58-66)-specific clones expressing non-Vbeta17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M1(58-66) complex could influence functional properties, M1(58-66)-specific clones expressing subdominant (non-Vbeta17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vbeta17+) TCR. The Vbeta17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vbeta gene segments. All Vbeta17+ CTL clones tested bound HLA-A*0201/M1(58-66) tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vbeta17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vbeta17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vbeta17+ CTL. The dominance of Vbeta17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M1(58-66) complex.

Functional heterogeneity and high frequencies of cytomegalovirus-specific CD8(+) T lymphocytes in healthy seropositive donors.

Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8(+) T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8(+) T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in "fresh" killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Direct visualization and quantitation of cytomegalovirus-specific CD8+ cytotoxic T-lymphocytes in liver transplant patients.

BACKGROUND: CMV infection remains a significant clinical problem in the context of LT. Changes in the magnitude of the CMV-specific CTL response after LT have not previously been assessed but may be important in determining the outcome of CMV infection. METHOD: We used a fluorescent HLA-B*0702-CMV peptide tetrameric complex to directly visualize and quantitate CMV-specific CD8+ CTL both in immunosuppressed patients after LT and in immunocompetent controls. RESULTS: CMV-specific CD8+ CTL, at a frequency ranging from 0.1 to 5.8% of CD8+, were detected in the peripheral blood of 22 of 25 B*0702, CMV immunoglobulin G seropositive individuals, with no difference observed between immunocompetent controls and patients >3 years after LT. In CMV seropositive LT recipients who did not have symptomatic CMV infection during the first 3 months after LT, CMV-specific CD8+ CTL magnitude initially decreased, then increased up to 5 times higher than pre-LT levels within 3 months. Two CMV seronegative recipients of seropositive donors had symptomatic CMV infection in association with high viral load. In both patients, no CD8+ CTL response was detected before the onset of symptoms, and a reduction in viral load was observed during antiviral therapy. However, polymerase chain reaction negativity was achieved only when a demonstrable CMV-specific CD8+ CTL response was generated. Responses were never observed in asymptomatic CMV seronegative patients. CONCLUSIONS: We suggest that the generation of CMV-specific CD8+ CTL may be driven by, and seems to coincide with the suppression of, viral reactivation. Direct monitoring of CMV-specific CD8+ CTL using an HLA-peptide tetramer may prove to be of value in the management of patients after LT.

Multiple T cell expansions are found in the blood and synovial fluid of patients with reactive arthritis.

OBJECTIVE: To look for evidence of T lymphocyte expansions in the blood and synovial fluid (SF) of patients with reactive arthritis (ReA). METHODS: Paired peripheral blood and synovial samples from 10 patients with ReA were studied by dual color flow cytometry using T cell receptor (TCR) V beta specific and CD4 or CD8 specific antibodies. Two synovial CD8 expansions were studied by 3 color flow cytometry. Peripheral blood samples from 13 healthy, age matched individuals were studied as controls. RESULTS: Statistically significant expansions were observed in all patients, occurring in blood and SF CD4 and CD8 compartments, but were most common in the synovial CD8 compartment. Expansions studied in further detail displayed an activated "memory" phenotype. A synovial BV22S1/CD8 expansion was seen in 5/6 patients with sexually acquired ReA. CONCLUSION: Multiple T lymphocyte expansions are found in both the blood and SF of patients with ReA. Expansions were most commonly found in the synovial CD8 compartment, where they appeared to express both activation and memory markers. This indicates that T lymphocytes (and in particular cytotoxic T cells) may play a pathogenic role in ReA. These findings are consistent with either an antigen or a superantigen driven response.

Large clonal expansions of CD8+ T cells in acute infectious mononucleosis.

Primary infection with Epstein-Barr virus often results in the clinical syndrome of acute infectious mononucleosis (glandular fever). This illness is characterized by a striking lymphocytosis, the nature of which has been controversial. We show that large monoclonal or oligoclonal populations of CD8+ T cells account for a significant proportion of the lymphocytosis and provide molecular evidence that these populations have been driven by antigen. The results suggest that the selective and massive expansion of a few dominant clones of CD8+ T cells is an important feature of the primary response to this virus.

Oral manifestations of HIV infection: a Panamerican perspective.

This paper presents a preliminary approach to the study of the oral manifestations of HIV infections in the region of the Americans. A general description of the lesions encountered is provided together with a review of the prevalence of the different manifestations in some countries of the Americas. Oral candidiasis was the most common oral lesion identified. Among oral candidiasis lesions differences were noted in relation to the frequency of the clinical forms seen. Hairy leukoplakia was the second most frequent lesion in almost all studies, with the exception the Peruvian study, where the most prevalent oral condition was xerostomia. The numbers of cases of HIV-gingivitis and HIV-periodontitis found in the countries of the Americas were lower than the cases in USA. Other oral manifestations of HIV infections seen were: Kaposi's sarcoma, oral erythema, labial herpetic infection. It is concluded that still more studies are needed, oral health professionals need additional training in the detection and treatment of lesions, and information needs to be systematized and standardized such that it is possible to make accurate comparisons among regions and countries. Recommendations are included to improve this situation.

PIP: The oral cavity is a common site for the occurrence of lesions in patients who are seropositive for HIV. This paper describes a preliminary approach to the study of the oral manifestations of HIV infections in the region of the Americas. Specifically, a general description of typical lesions along with a review of the prevalence of different manifestations is presented based on data from Argentina, Brazil, Chile, Mexico, Peru, US, Uruguay, and the English Caribbean. Although differences were noted in the frequency of the clinical forms seen, oral candidiasis was the most common oral lesion identified. Hairy leukoplakia was the second most frequent lesion in all studies except the Peruvian in which the most prevalent oral condition was xerostomia. Fewer cases of HIV-gingivitis and HIV-periodontitis were seen in the Americas than in the US. Other manifestations of HIV infections observed include Kaposi's sarcoma, oral erythema, and labial herpetic infection. More studies are needed; dentists need more training in detecting and treating lesions; and information needs to be systematized and standardized so that accurate comparisons may be made among regions and countries.

Subgingival microflora of advanced periodontitis in the Dominican Republic.

A study of the predominant subgingival microflora was carried out in 24 periodontitis patients, 18 to 60 years of age, in Santo Domingo, Dominican Republic. Paper point sampling, transport in VMGA III, and conventional microbiological techniques were utilized. Direct microscopic examination revealed that cocci and nonmotile organisms made up 85% of the total organisms and spirochetes as little as 3%. Nonselective culturing showed Gram-negative organisms to constitute 53% of total isolates. Fusobacterium nucleatum averaged 15%, black-pigmented anaerobes 7%, and Peptostreptococcus micros 10% of the cultivable microflora. Enteric rods and acinetobacter species were recovered from 16 patients and comprised 23% of the cultivable flora. Enterobacter cloacae occurred in 8 patients, Klebsiella oxytoca in 3 patients, and 7 other species in 10 patients. Parallel studies have found a significantly lower prevalence of enteric rods in advanced periodontitis patients in the USA. In conclusion, fewer spirochetes and markedly more enteric rods seem to inhabit adult periodontitis lesions in Santo Domingo patients compared to those in USA. High levels of subgingival enteric rods in periodontitis patients in Santo Domingo may have important prophylactic and therapeutic implications.

Governmental expectations of dentistry.

The interests of governments in dental health are recognized but not always best served in communication with the dental profession. Nor does the profession communicate readily with the public. In seeking to influence governmental policies, there is a need to collect and interpret data on dental disease in forms which will make impact within the governmental framework and lead to more rapid action. Greater public awareness will help in the formulation of government policies but only if dictates for action are supplemented by explanations of their aims. Dentistry must be more prepared to listen to government and to approach the solution of common problems in a constructive and flexible manner. In doing this dentistry must aim to provide services to which all will have access.

[Innovative trends in dentistry: background, bases and outlook]. / Tendencias innovadoras en odontología: antecedentes, bases y perspectivas.

This article briefly analyzes the present situation in dental practice with emphasis on its inherent discrepancies, and describes the background, theoretic and methodologic bases, and prospects of the PAHO/WHO Program of Innovation in Dental Education and Care. An account is given of the conceptual framework of the Program, which is based on the hypothesis that changes in oral health in a country depend partly on the national socioeconomic situation at any given point in its history. At the same time, it is recognized that there is leeway in the general political setting for the trial and application of plans for curricula and care that can work quantitative and qualitative changes in the educational and service sectors. There follows a description of the operational arrangement and the subprograms and prpkects going forward in 16 dental schools of Latin America.

Enamel hypoplasia in relation to caries in Guatemalan children.

Guatemalan children with anterior linear enamel hypoplasia (LEH) had a significantly greater caries experience in posterior dentition than their peers who did not have anterior LEH. The findings suggest that the synergistic mechanism of undernutrition and infection, which may underlie the occurrence of anterior LEH, may also predispose clinically normal appearing deciduous molars to an excessive caries attack equal to that observed in the grossly hypoplastic anterior teeth. The nutritional implications merit further investigation.