Naturally Arising Human CD4 T-Cells That Recognize Islet Autoantigens and Secrete Interleukin-10 Regulate Proinflammatory T-Cell Responses via Linked Suppression

OBJECTIVE—Regulatory T-cells (Tregs) recognizing islet au-toantigens are proposed as a key mechanism in the maintenance of self-tolerance and protection from type 1 diabetes. To date, however, detailed information on such cells in humans, and insight into their mechanisms of action, has been lacking. We previously reported that a subset of CD4 T-cells secreting high levels of the immunosuppressive cytokine interleukin-10 (IL-10) is significantly associated with late onset of type 1 diabetes and is constitutively present in a majority of nondiabetic individuals. Here, we test the hypothesis that these T-cells represent a naturally generated population of Tregs capable of suppressing proinflammatory T-cell responses. RESEARCH DESIGN AND METHODS—We isolated and cloned islet-specific IL-10–secreting CD4 T-cells from nondia-betic individuals after brief ex vivo exposure to islet autoantigen

Combinatorial detection of autoreactive CD8+ T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop

Aims/hypothesis: Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8+ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting. Methods: The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers. Results: The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8+ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8+ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors. Conclusions/interpretation Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting

Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell–Associated Epitopes Using Combinatorial MHC Multimers

textabstractOBJECTIVE - Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope-specific CD8+T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8+T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS - Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10-18, prepro-insulin (PPI)15-24, islet antigen (IA)-2797-805, GAD65114-123, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)265-273, and pre-pro islet amyloid polypeptide (ppIAPP)5-13-specific CD8+T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS - Using this kit, islet autoreactive CD8+T-cells recognizing insulin B10-18, IA-2797-805, and IGRP265-273were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15-24proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS - A kit was developed that allows simultaneous detection of CD8+T-cells reactive to multiple HLA-A2-restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples

Plasmacytoid Dendritic Cells Are Proportionally Expanded at Diagnosis of Type 1 Diabetes and Enhance Islet Autoantigen Presentation to T-Cells Through Immune Complex Capture

OBJECTIVE—Immune-mediated destruction of β-cells resulting in type 1 diabetes involves activation of proinflammatory, islet autoreactive T-cells, a process under the control of dendritic cells of the innate immune system. We tested the hypothesis that type 1 diabetes development is associated with disturbance of blood dendritic cell subsets that could enhance islet-specific autoimmunity

Plasma cytokines in women with chronic fatigue syndrome

© 2009 Fletcher et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Air temperature changes in Toruń (central Poland) from 1871 to 2010

The article presents a detailed analysis of changes in air temperature in Toruń in the period 1871–2010 on the basis of homogenised monthly, seasonal and annual air temperature series which have been newly constructed (i.e. extended by the 50 years of 1871–1920). Over the 140-year study period, a sizeable and statistically significant increase of 0.1 °C per decade was found in the air temperature in Toruń. The greatest increases occurred for spring and winter, at 0.12 and 0.11 °C, respectively. A lesser warming, meanwhile, was recorded for autumn (0.10 °C/10 years), and particularly for summer (0.07 °C/10 years). The air temperature trends are statistically significant for all seasons. Air temperature differences between the monthly averages of three analysed subperiods (1871–1900, 1901–1950 and 1951–2010) and averages for the entire period under review rarely exceeded ± 0.5 °C. In all of these periods, the highest average air temperatures occurred in July and the lowest in January. The period of 1981–2010 had the highest frequency of occurrence of very and extremely warm seasons and years. Meanwhile, the highest frequency of very and extremely cool seasons and years was recorded in the 1940s and in the nineteenth century. In the period of 1871–2010, winters shortened markedly (by 7%) and summers lengthened by 3.8%. All of the presented aspects of air temperature in Toruń, which is representative of the climate of central Poland, are in close agreement with the findings of analogous studies of the same for other areas of Poland and Central Europe

Islet-Specific CTL Cloned from a Type 1 Diabetes Patient Cause Beta-Cell Destruction after Engraftment into HLAA2 Transgenic NOD/SCID/IL2RG Null Mice

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP(265-273)-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγ(null) mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies

Type 1 diabetes: translating mechanistic observations into effective clinical outcomes

Type 1 diabetes remains an important health problem, particularly in Western countries where the incidence has been increasing in younger children(1). In 1986, Eisenbarth described Type 1 diabetes as a chronic autoimmune disease. Work over the past 3 ½ decades has identified many of the genetic, immunologic, and environmental factors that are involved in the disease and have led to hypotheses concerning its pathogenesis. Based on these findings, clinical trials have been conducted to test these hypotheses but have had mixed results. In this review, we discuss the findings that have led to current concepts of the disease mechanisms, how this understanding has prompted clinical studies, and the results of these studies. The findings from preclinical and clinical studies support the original proposed model for how type 1 diabetes develops, but have also suggested that this disease is more complex than originally thought and will require broader treatment approaches

Relief and land use impact on the atmospheric precipitation structure in Cracow's surroundings (1971-2005)

W pracy zaprezentowano zróżnicowanie czasowe i przestrzenne sum miesięcznych i rocznych opadów atmosferycznych na trzech stacjach położonych w okolicach Krakowa w przekroju południkowym: Garlicy Murowanej, Krakowie – Ogrodzie Botanicznym i Gaiku-Brzezowej w latach 1971–2005. Latem opady w Gaiku-Brzezowej były o około 20% wyższe, a zimą opady w Garlicy o ok. 25% niższe niż na pozostałych dwóch stacjach. Najwyższa średnia roczna suma opadów charakteryzowała stację w Gaiku: 747,5 mm (tab. 1). Największa zmienność charakteryzuje opady w Garlicy, szczególnie w miesiącach półrocza chłodnego (rys. 1). Najwyższe roczne sumy opadów zanotowano w 2001 r. w Garlicy (865,2 mm) i Krakowie (845,9 mm) i w 1974 r. w Gaiku (1178,7 mm), natomiast najniższe sumy wystąpiły wszędzie w 1993 r. (odpowiednio 446,1, 482 i 524,8 mm). Stwierdzono znaczne zróżnicowanie opadów z roku na rok i dużą nieregularność ich występowania oraz brak wyraźnej tendencji zmian (tab. 2, rys. 2). Średnia roczna liczba dni z opadem wyniosła 132 dni w Garlicy, 168 w Gaiku i 216,4 w Krakowie, ale w przypadku dni z sumą dobową ≥5 mm ich liczba dla Garlicy i Krakowa jest prawie taka sama, natomiast nieco wyższe wartości charakteryzują Gaik. Najwięcej dni z opadami mało wydajnymi jest w Krakowie, natomiast największa liczba dni z wysokimi sumami opadów charakteryzuje Gaik (tab. 3). Każda z rozpatrywanych stacji ma odrębne warunki opadowe: w Garlicy opady są najniższe i o największej zmienności, stacja miejska w Krakowie charakteryzuje się największą liczbą dni z dobową sumą opadów 1–5 mm, w Gaiku zaś roczne i miesięczne sumy opadów są zazwyczaj największe i występuje najwięcej dni z dobową sumą opadów powyżej 10 mm. Pogórze Wielickie ma znacznie korzystniejsze warunki opadowe niż pd. skłon Wyżyny Krakowsko-Częstochowskiej z uwagi na wyższe sumy miesięczne i roczne opadów oraz mniejszą ich zmienność.The paper presents temporal and spatial differentiation of monthly and annual sums of atmospheric precipitation at three stations located in Cracow’s surroundings along a longitudinal profile: Garlica Murowana (representing the Cracow- Częstochowa Upland), Cracow-Botanical Garden (in the Vistula river valley) and Gaik-Brzezowa (in the Wieliczka Foothills, a part of the Carpathian Foothills), in the years 1971-2005. In summer, the precipitation was by 20% higher in Gaik-Brzezowa and in winter by 25% lower in Garlica than at other two stations. The highest mean annual precipitation sum was characteristic for Gaik: 747.5 mm (Table 1). The largest variability is typical for the precipitation in Garlica, especially in cold half-year (Figure 1). The highest annual precipitation sums were noted in 2001 in Garlica (865.2 mm) and Cracow (845.9 mm) and in 1974 in Gaik (1178.7 mm), while the lowest annual sums occurred everywhere in 1993 (446.1, 482 and 524.8 mm, respectively). The precipitation sums show large inter-annual variability, high irregularity and lack of any clear tendency (Table 2, Figure 2). Mean annual number of days with precipitation reached 132 mm in Garlica, 168 mm in Gaik and 216.4 mm in Cracow but in case of the days with daily precipitation sum ≥5 mm their numbert for Garlica and Cracow is almost the same while a bit higher values can be found for Gaik. The highest number of days with low precipitation sums is characteristic for Cracow, while the highest number of days with high precipitation sums occurs in Gaik (Table 3). Each of the stations studied has distinguished precipitation conditions: Garlica is the driest point and with the highest precipitation variability, urban station in Cracow is characterised by the highest number of days with daily precipitation sum 1-5 mm, and in Gaik the annual and monthly precipitation sums are usually the highest and there is the highest number of days with daily precipitation sum above 10 mm. The Wieliczka Foothills has much better precipitation conditions for agriculture than the southern slope of the Cracow-Częstochowa Upland due to higher monthly and annual precipitation sums and their lower variability

The influence of synoptic situations on air temperature in the south part of the Krakowsko-Czestochowska Upland

Przedmiotem podjętych badań były warunki termiczne w połudnowej części Wyżyny Krakowsko-Częstochowskiej. Uznano za interesujące przeanalizowanie wpływu sytuacji synoptycznych na wartości odchyleń temperatury powietrza od średniej wieloletniej. Cyrkulacja atmosferyczna w porównaniu do czynników geograficznych jest procesem bardzo zmiennym w czasie. Stąd też, tak ważne znaczenie odgrywają badania zmierzające do ilościowego określenia zróznicowania elementów klimatu przy panujących sytuacjach synoptycznych. W badaniach wykorzystano typy sytuacji synoptycznych dla Dorzecza Górnej Wisły opracowane przez T. Niedźwiedzia oraz wyniki obserwacji temperatury powietrza z lat 1991–2005 ze stacji meteorologicznych reprezentujących odmienne formy terenu. Formy płaskie reprezentuje płaszczyzna w Krakowie- Balicach, wklęsłe dno doliny w Ojcowie, natomiast wypukłe wierzchowina położona w Garlicy Murowanej. W wyniku przeprowadzonych badań stwierdzono, że na analizowanym obszarze częściej pogodę kształtowały sytuacje antycyklonalne (56%) niż cyklonalne (44%). Najczęściej występowały typy sytuacji: Ka (13,8%), Bc(11,8%), Wa(9,5%) i Wc(8,7%). Najrzadziej zaś notowano sytuacje Ec(1.3%) i Cc(0,7%) oraz X (1,7%) i NEc(1,8%). Jak wykazały badania, we wszystkich porach roku występowało duże zróżnicowanie odchyleń temperatury zarówno co do wartości bezwzględnej jak i znaku.The object of the study was the thermal conditions in the southern part of the Krakowsko-Częstochowska Upland. The analysis of the influence of synoptic situations on the value of air temperature deviation from perennial mean. Recent studies more often analyze standard meteorological elements as well as synoptic situations which are characterized by specific spacious pressure distribution and the direction of air mass. Atmospheric circulation in comparison to geographical factors of climate is a process variable in time. Therefore the studies which aim to describe differentiation of the climate elements in current synoptic situations are so important. The types of synoptic situations described by T. Niedzwiedz were used for the Upper Vistula River Basin. Also the results of the air temperature observations carried from 1991 to 2005 at the meteorological stations in Kraków-Balice, Garlica Murowana and Ojców. The stations represent the most typical forms of the area i.e. flattened valley (Kraków-Balice), hill (Garlica Murowana) and deep valley (Ojców). The research showed that in the study area anticyclonic situations (56%) occurred more often than cyclonic situations (44%). The most frequent situations were: Ka (13,%), Bc (11,8%), Wa (9,5%) and Wc (8,7%). The least frequent were: Ec (1,3%), Cc (0,7%) and X (1,7%) and NEc (1,8%). As the research showed the deviation of mean and extreme temperature at the stations was characterized by high differentiation of absolute value during all the seasons of the year