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Abstract Half of patients with renal cell carcinoma (RCC) develop metastases. New and noninvasive biomarkers are needed for the diagnosis of RCC. The study aims to develop an EV‐based assay for the detection of RCC using a highly sensitive nanoparticle‐aided time‐resolved fluorescence immunoassay (NP‐TRFIA). To confirm the presence of tetraspanins on EVs, size exclusion chromatography is used to separate EV‐ and PE‐fractions from RCC4, 786‐O, and HEK293 cell lines. EV‐ and PE‐fractions are quantified using NP‐TRFIA assays established for CD9, CD63, CD81, and CD151. Tetraspanins are measured from RCC CCM and serum samples of RCC (n = 14), benign (n = 17), and healthy (n = 9) individuals. Among the tetraspanins, CD63 exhibits 3‐5‐fold higher expression on RCC4 and 786‐O CCM compared to HEK293. A sandwich CD63‐CD63 assay demonstrates significant discrimination of RCC patients from benign (p = 0.0003), and healthy (p = 0.005) individuals, respectively. Similarly, the CD81‐CD81 assay also enables significant separation of RCC patients compared to benign (p = 0.014), and healthy (p = 0.003) controls, respectively. This suggests that RCC cell lines and serum of RCC patients show higher amounts of CD63‐ and CD81‐EVs compared to controls. Detection of these EVs using NP‐TRFIA approach may play a vital role in the detection of RCC.

Abstract After surgery of localized renal cell carcinoma, over 20% of the patients will develop distant metastases. Our aim was to develop an easy-to-use prognostic model for predicting metastasis-free survival after radical or partial nephrectomy of localized clear cell RCC. Model training was performed on 196 patients. Right-censored metastasis-free survival was analysed using LASSO-regularized Cox regression, which identified three key prediction features. The model was validated in an external cohort of 714 patients. 55 (28%) and 134 (19%) patients developed distant metastases during the median postoperative follow-up of 6.3 years (interquartile range 3.4–8.6) and 5.4 years (4.0–7.6) in the training and validation cohort, respectively. Patients were stratified into clinically meaningful risk categories using only three features: tumor size, tumor grade and microvascular invasion, and a representative nomogram and a visual prediction surface were constructed using these features in Cox proportional hazards model. Concordance indices in the training and validation cohorts were 0.755 ± 0.029 and 0.836 ± 0.015 for our novel model, which were comparable to the C-indices of the original Leibovich prediction model (0.734 ± 0.035 and 0.848 ± 0.017, respectively). Thus, the presented model retains high accuracy while requiring only three features that are routinely collected and widely available.

AbstractObjective To evaluate the association between childhood parental smoking exposure and the risk of overweight/obesity from childhood to adulthood.Methods This study leverages the data from two longitudinal population based cohort studies, the Cardiovascular Risk in Young Finns Study between years 1980–2011/2012 (YFS; N = 2,303; baseline age 3–18 years) and the Special Turku Coronary Risk Factor Intervention Project between years 1989–2009/2010 (STRIP; N = 632; baseline age 7 months). Weight, height and waist circumference were measured from childhood to adulthood. Overweight/obesity was defined as body mass index ≥25 kg/m2 in adults and using the Cole criteria in children. Central obesity was defined as waist circumference > 100/90 cm in men/women and as a waist-to-height ratio > 0.50 in children. Statistical analyses were adjusted for age, sex, socioeconomic status, smoking, birth weight, parental ages, diet and physical activity.Results Childhood parental smoking exposure was associated with increased risk for life-course overweight/obesity (YFS: RR1.13, 95%CI 1.02–1.24; STRIP: RR1.57, 95%CI 1.10–2.26) and central obesity (YFS: RR1.18, 95%CI 1.01–1.38; STRIP: RR1.45, 95%CI 0.98–2.15).Conclusions Childhood exposure to parental smoking is associated with increased risk of overweight/obesity over the life-course.KEY MESSAGESExposure to parental smoking in childhood was associated with increased risk of overweight/obesity, central obesity and adiposity measured by skinfold thickness from childhood to adulthood.

Abstract Background Cells in solid tumours are variably hypoxic and hence resistant to radiotherapy - the essential role of oxygen in the efficiency of irradiation has been acknowledged for decades. However, the currently available methods for performing hypoxic experiments in vitro have several limitations, such as a limited amount of parallel experiments, incapability of keeping stable growth conditions and dependence on CO2 incubator or a hypoxia workstation. The purpose of this study was to evaluate the usability of a novel portable system (Minihypoxy) in performing in vitro irradiation studies under hypoxia, and present supporting biological data. Materials and methods This study was conducted on cancer cell cultures in vitro. The cells were cultured in normoxic (~ 21% O2) or in hypoxic (1% O2) conditions either in conventional hypoxia workstation or in the Minihypoxy system and irradiated at dose rate 1.28 Gy/min ± 2.9%. The control samples were sham irradiated. To study the effects of hypoxia and irradiation on cell viability and DNA damage, western blotting, immunostainings and clonogenic assay were used. The oxygen level, pH, evaporation rate and osmolarity of the culturing media on cell cultures in different conditions were followed. Results The oxygen concentration in interest (5, 1 or 0% O2) was maintained inside the individual culturing chambers of the Minihypoxy system also during the irradiation. The radiosensitivity of the cells cultured in Minihypoxy chambers was declined measured as lower phosphorylation rate of H2A.X and increased clonogenic capacity compared to controls (OER~ 3). Conclusions The Minihypoxy system allows continuous control of hypoxic environment in multiple wells and is transportable. Furthermore, the system maintains the low oxygen environment inside the individual culturing chambers during the transportation and irradiation in experiments which are typically conducted in separate facilities.

Abstract Background A key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-α, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not clear. Methods To gain insight into the functions of high PHD3 expression in ccRCC, we used PHD3 knockdown by siRNA in 786-O cells under normoxic and hypoxic conditions and performed discovery mass spectrometry (LC-MS/MS) of the purified peptide samples. The LC-MS/MS results were analysed by label-free quantification of proteome data using a peptide-level expression-change averaging procedure and subsequent gene ontology enrichment analysis. Results Our data reveals an intriguingly widespread effect of PHD3 knockdown with 91 significantly regulated proteins. Under hypoxia, the response to PHD3 silencing was wider than under normoxia illustrated by both the number of regulated proteins and by the range of protein expression levels. The main cellular functions regulated by PHD3 expression were glucose metabolism, protein translation and messenger RNA (mRNA) processing. PHD3 silencing led to downregulation of most glycolytic enzymes from glucose transport to lactate production supported by the reduction in extracellular acidification and lactate production and increase in cellular oxygen consumption rate. Moreover, upregulation of mRNA processing-related proteins and alteration in a number of ribosomal proteins was seen as a response to PHD3 silencing. Further studies on upstream effectors of the translational machinery revealed a possible role for PHD3 in regulation of mTOR pathway signalling. Conclusions Our findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.

Purpose: TILT-123 (igrelimogene litadenorepvec) is an oncolytic adenovirus armed with TNFa and IL2, designed to induce T-cell infiltration and cytotoxicity in solid tumors., Patients and Methods: TUNIMO (NCT04695327) was a single-arm, multicenter phase I dose-escalation trial designed to assess the safety of TILT-123 in advanced solid cancers refractory to standard therapy. Patients received intravenous and intratumoral TILT-123. The primary endpoint was safety by adverse events (AE), laboratory values, vital signs, and electrocardiograms. Secondary endpoints included tumor response, pharmacokinetics, and predictive biomarkers., Results: Twenty patients were enrolled, with a median age of 58 years. Most prevalent cancer types included sarcomas (35%), melanomas (15%) and ovarian cancers (15%). No dose-limiting toxicities were observed. The most frequent treatment-related AEs included fever (16.7%), chills (13.0%), and fatigue (9.3%). Ten patients were evaluable for response on day 78 with RECIST 1.1, iRECIST or PET-based evaluation. The disease control rate by PET was 6/10 (60% of evaluable patients) and 2/10 by RECIST 1.1 and iRECIST(20%of evaluable patients). Tumor size reductions occurred in both injected and non-injected lesions. TILT-123 was detected in injected and non-injected tumors, and virus was observed in blood after intravenous and intratumoral injections. Treatment resulted in reduction of lymphocytes in blood, with concurrent lymphocyte increases in tumors, findings compatible with trafficking., Conclusions: TILT-123 was safe and able to produce antitumor effects in local and distant lesions in heavily pre-treated patients. Good tolerability of TILT-123 facilitates combination studies, several of which are ongoing (NCT04217473, NCT05271318, NCT05222932, and NCT06125197). See related commentary by Silva-Pilipich and Smerdou, p. 3649., (©2024 The Authors; Published by the American Association for Cancer Research.)

The percentage of obese people is increasing worldwide, causing versatile health problems. Obesity is connected to diseases such as diabetes and cardiovascular diseases, which are preceded by a state called metabolic syndrome. Diets rich in fruits and vegetables have been reported to decrease the risk of metabolic syndrome and type 2 diabetes. Berries with a high polyphenol content, including lingonberry ( Vaccinium vitis - idaea L.), have also been of interest to possibly prevent obesity-induced metabolic disturbances. In the present study, we prepared an extract from the by-product of a lingonberry juice production process (press cake/pomace) and investigated its metabolic effects in the high-fat diet-induced model of obesity in mice. The lingonberry skin extract partly prevented weight and epididymal fat gain as well as a rise in fasting glucose level in high-fat diet-fed mice. The extract also attenuated high-fat diet-induced glucose intolerance as measured by an intraperitoneal glucose tolerance test (IPGTT). The extract had no effect on the levels of cholesterol, triglyceride or the adipokines adiponectin, leptin, or resistin. The results extend previous data on the beneficial metabolic effects of lingonberry. Further research is needed to explore the mechanisms behind these effects and to develop further health-promoting lingonberry applications.

Objective: Immune checkpoint inhibitors (ICI) can cause endocrine adverse events. However, endocrine adverse events (AEs). However, endocrine AEs could be related to better treatment outcomes. Our aim was to investigate whether this holds true in a real-world setting of metastatic melanoma patients., Design: A retrospective single-institution study., Methods: We included 140 consecutive metastatic melanoma patients treated with ICI between January 2012 and May 2019. We assessed the endocrine toxicity and the best possible treatment outcomes from electronic patient records, including laboratory parameters and radiological images., Results: Of the treated patients, 21 patients (15%) were treated with ipilimumab, 46 (33%) with nivolumab, 67 (48%) with pembrolizumab, and 6 (4%) with combination therapy (ipilimumab + nivolumab). Endocrine AEs appeared in 29% (41/140) patients. Three patients had two different endocrine AEs. Thyroid disorders were the most common: 26% (36/140), followed by hypophysitis: 4% (5/140). Three subjects (2%, 3/140) were diagnosed with autoimmune diabetes. Three patients had to terminate treatment due to endocrine toxicity. Radiological manifestations of endocrine AEs were found in 16 patients (39%, 16/41). Endocrine toxicity was associated with significantly better treatment outcomes. Median progression-free survival (8.1 months, range 5.1-11.1 months vs 2.7 months, range 2.4-3.0 months, P < 0.001), and median overall survival (47.5 months, range 15.5-79.5 months vs 23.7 months, range 15.3-32.1 months, P = 0.035) were longer for patients experiencing endocrine AEs., Conclusions: The higher number of endocrine AEs suggest that regular laboratory monitoring aids in AE detection. Endocrine AEs in metastatic melanoma may correlate with better treatment outcomes.

Although new compounds have improved the treatment landscape of metastatic melanoma, very limited data exist on the efficacy and safety of treating older patients with novel agents. Here, we provide results of BRAF (BRAFi) ± MEK (MEKi) inhibitor treatment in patients over 75 years (oldest-old patients) with metastatic melanoma. Between 2011 and 2020, 34 consecutive patients with metastatic melanoma over 75 years of age (range 75-89) were treated with BRAFi ± MEKi at the Comprehensive Cancer Center of Helsinki University Hospital. Data on clinical and histopathological features, toxicity, response rate (RR), progression-free survival (PFS) and overall survival (OS) were collected. Patients were treated with BRAFi (n = 22) or BRAFi in combination with MEK inhibitor (MEKi) (n = 12). Grade 1-2 adverse events occurred in 68% of the patients, 32% had grade 3 adverse effects, dose reductions were made for 41% of patients and 29% terminated treatment due to toxicity. Overall, the RR was 62%. Complete responses were achieved in 27% of the patients, and 35% had partial responses. The median PFS was 8 months (range 0-57), and the median OS was 15 months (range 0-71). Tailored BRAFi ± MEKi treatment for older patients is feasible. Adverse effects occur frequently but are manageable by dose adjustment. The occurrence of toxicity of monotherapy was similar to that of combination therapy. The RR and median OS from our retrospective study are comparable with those reported in clinical trials and combination therapy produced somewhat more and longer-lasting responses. Hence, it seems that older patients may benefit from BRAFi treatment., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The microbes present in bioethanol production processes have been previously studied in laboratory-scale experiments, but there is a lack of information on full-scale industrial processes. In this study, the microbial communities of three industrial bioethanol production processes were characterized using several methods. The samples originated from second-generation bioethanol plants that produce fuel ethanol from biowaste, food industry side streams, or sawdust. Amplicon sequencing targeting bacteria, archaea, and fungi was used to explore the microbes present in biofuel production and anaerobic digestion of wastewater and sludge. Biofilm-forming lactic acid bacteria and wild yeasts were identified in fermentation samples of a full-scale plant that uses biowaste as feedstock. During the 20-month monitoring period, the anaerobic digester adapted to the bioethanol process waste with a shift in methanogen profile indicating acclimatization to high concentrations of ammonia. Amplicon sequencing does not specifically target living microbes. The same is true for indirect parameters, such as low pH, metabolites, or genes of lactic acid bacteria. Since rapid identification of living microbes would be indispensable for process management, a commercial method was tested that detects them by measuring the rRNA of selected microbial groups. Small-scale testing indicated that the method gives results comparable with plate counts and microscopic counting, especially for bacterial quantification. The applicability of the method was verified in an industrial bioethanol plant, inspecting the clean-in-place process quality and detecting viability during yeast separation. The results supported it as a fast and promising tool for monitoring microbes throughout industrial bioethanol processes.

Numerous studies have shown evidence supporting the benefits of universal newborn screening for primary immunodeficiencies (PID) and for Spinal Muscular Atrophy (SMA). We have developed a four-plex, real-time PCR assay to screen for Severe Combined Immune Deficiencies (SCID), X-linked agammaglobulinemia (XLA), and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS). A simple, high-throughput, semi-automated DNA extraction method was developed for a Janus liquid handler that can process 384 DBS punches in four 96-well plates in just over one hour with sample tracking capability. The PCR assay identifies the absence of exon 7 in the SMN1 gene, while simultaneously evaluating the copy number of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules. Additionally, the amplification of a reference gene, RPP30 , was included in the assay as a quality/quantity indicator of DNA isolated from the DBS. The assay performance was demonstrated on over 3000 DNA samples isolated from punches of putative normal newborn DBS. The reliability and analytical accuracy were further evaluated using DBS controls, and contrived and confirmed positive samples. The results from this study demonstrate the potential of future molecular DBS assays, and highlight how a multiplex assay could benefit newborn screening programs., Competing Interests: Conflicts of InterestC.G-M., A.T., K.V., M.J., G.F., S.D., D.G., D.S. and R.W. are PerkinElmer employees. Authors representing PerkinElmer had a role in designing the study, in collection, analyses, and interpretation of the data, in the writing of the manuscript, and in the decision to publish the results. The other authors declare no conflict of interest., (© 2019 by the authors.)

The catalytic conversion of lignocellulosic biomass is attractive due to the feasible generation of valuable products such as reducing sugars which constitute the basic substrates for chemical and transportation fuel production, as well as the production of renewable hydrogen. This study shows the efficient conversion of lignocellulose, especially hemicellulose, into reducing sugars such as xylose and galactose, by mechanocatalysis using potassium pyrosulfate, K 2 S 2 O 7 , as an effective salt catalyst. Ball milling was performed, introducing a mechanical force which, combined with chemical pretreatment, leads to reducing sugar yields (40%) almost as high as when commonly used sulfuric acid was employed. Kinetic experiments as well as the optimization of the saccharification process are presented., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Quinoa is a crop that originated from the Andes. It has high nutritional value, outstanding agro-ecological adaptability, and low water requirements. Quinoa is an excellent crop alternative to help overcome food shortages, and it can also have a role in the prevention of developed world lifestyle diseases, such as type-2 diabetes, cardiovascular diseases, osteoporosis, inflammatory and autoimmune diseases, etc. In order to expand the traditional uses of quinoa and to provide new, healthier and more nutritious food products, a fermented quinoa-based beverage was developed. Two quinoa varieties (Rosada de Huancayo and Pasankalla) were studied. The fermentation process, viscosity, acidity, and metabolic activity during the preparation and storage of the drink were monitored, as well as the preliminary organoleptic acceptability of the product. The drink had viable and stable microbiota during the storage time and the fermentation proved to be mostly homolactic. Both quinoa varieties were suitable as base for fermented products; Pasankalla, however, has the advantage due to higher protein content, lower saponin concentration, and lower loss of viscosity during the fermentation process. These results suggest that the differences between quinoa varieties may have substantial effects on food processes and on the properties of final products. This is a factor that should be taken into account when planning novel products based on this grain.

This study describes a time and energy saving, solvent-free procedure for the conversion of lignocellulosic barley straw into reducing sugars by mechanocatalytical pretreatment. The catalytic conversion efficiency of several solid acids was tested which revealed oxalic acid dihydrate as a potential catalyst with high conversion rate. Samples were mechanically treated by ball milling and subsequently hydrolyzed at different temperatures. The parameters of the mechanical treatment were optimized in order to obtain sufficient amount of total reducing sugar (TRS) which was determined following the DNS assay. Additionally, capillary electrophoresis (CE) and Fourier transform infrared spectrometry (FT-IR) were carried out. Under optimal conditions TRS 42% was released using oxalic acid dihydrate as a catalyst. This study revealed that the acid strength plays an important role in the depolymerization of barley straw and in addition, showed, that the oxalic acid-catalyzed reaction generates low level of the degradation product 5-hydroxymethylfurfural (HMF)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.

The biological effects of polyphenolic ellagitannins are mediated by their intestinal metabolites, urolithins. This study investigated redox properties of urolithins A and B using ORAC assay, three cell-based assays, copper-initiated pro-oxidant activity (CIPA) assay, and cyclic voltammetry. Urolithins were strong antioxidants in the ORAC assay, but mostly pro-oxidants in cell-based assays, although urolithin A was an antioxidant in cell culture medium. Parent compound ellagic acid was a strong extracellular antioxidant, but showed no response in the intracellular assay. The CIPA assay confirmed the pro-oxidant activity of ellagitannin metabolites. In the cell proliferation assay, urolithins but not ellagic acid decreased growth and metabolism of HepG2 liver cells. In cyclic voltammetry, the oxidation of urolithin A was partly reversible, but that of urolithin B was irreversible. These results illustrate how strongly measured redox properties depend on the employed assay system and conditions and emphasize the importance of studying pro-oxidant and antioxidant activities in parallel.

Quercetin is a natural flavonoid possessing a number of health beneficial effects. Its bioactivity is restricted by low solubility and sensitivity to oxidative degradation, factors that are often ignored in laboratory studies. We studied the antimicrobial effects of quercetin on Staphylococcus aureus, Escherichia coli and Lactobacillus plantarum at concentrations at which it is soluble and investigated how the antioxidant vitamin C modulates these activities. S. aureus was the most sensitive of the studied bacteria. After 12 hours of culturing, 90 µM quercetin decreased the growth of S. aureus to 75 % of the value for a control culture. 1 mM vitamin C combined with 90 µM quercetin diminished the growth of S. aureus drastically to 3 % of that of the control culture supplemented with vitamin C only. Interestingly, vitamin C by itself inhibited the growth of S. aureus as well, and 5 mM vitamin C inhibited growth completely. The growth inhibition of E. coli was slightly but significantly better in the presence of both quercetin and vitamin C than in the presence of quercetin alone. Probiotic L. plantarum was resistant to quercetin in the presence and absence of vitamin C. Enhancement of quercetin's antimicrobial activity by vitamin C is partly explained by the stabilizing effect of vitamin C on quercetin. Even though the acidity of vitamin C contributes to the inhibition of S. aureus growth, neutralized vitamin C also inhibits the growth efficiently even without quercetin. Our results suggest that vitamin C affects the metabolism of S. aureus and that these changes are likely to result in the observed growth inhibition. Although vitamin C itself is a powerful antioxidant, its aerobic metabolism increases oxidative stress on bacterial cells. Vitamin C may therefore be a safe and natural alternative for restricting the growth of S. aureus when non-toxicity is required., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Cloudberry (Rubus chamaemorus L.) is one of the most valuable berry-producing plants because of its nutritional properties. The chemical composition and crop yield of ripe fruits of cloudberry grown wild in 10 habitats in northern Finland was analysed over two consecutive summers. For comparison, two clones of cultivated cloudberries were studied as well., Results: The concentrations of citric and malic acids, α-tocopherol, anthocyanins and β-carotene had notable variations between habitats. In particularly, cloudberries grown on open habitats had higher content of citric acid and less α-tocopherol compared to those grown on shaded sites. In a colder and rainy summer the content of anthocyanins and the unsaturation level of fatty acids were significantly higher than in a warmer and drier summer. Crop yields were higher in the warmer summer, except in open sites where yields were quite equal., Conclusion: Cloudberries grown in open habitats showed notable differences in chemical composition when compared to those grown on shaded sites. Results suggest that the amount of sunlight and temperature levels could be the main factors affecting crop yield and chemical composition of cloudberry. In addition, the amount of rainfall may have an effect on anthocyanin concentrations., (Copyright © 2011 Society of Chemical Industry.)

The study examined the validity of the Five to Fifteen (FTF) questionnaire in detecting developmental disorders in five-year-old children. A pilot study (N=70) was first undertaken to try out a translation of the FTF into Finnish. Subsequently, FTF questionnaires were mailed to parents of all five-year-old children from specified health-care districts in Vantaa, Finland. A total of 769 questionnaires (60%) were completed and returned. Children scoring above a cut-off value in any domain were considered at risk for having developmental disorders (the Risk Group; N=90). A Control Group was formed by randomly selecting 30 children who did not score above the threshold value in any domain. The children from the Risk Group and the Control Group were called to individual neuropsychological assessments. All invited children attended the assessments. The parts of the FTF that assess fine motor skills, executive functions (including attention and impulsivity), perception, memory, and language were used for this study. The external criterion measure was the NEPSY, a neuropsychological assessment instrument. Results demonstrated that the five FTF Domain Scores used in this study correlated significantly with the corresponding NEPSY Domain Scores. Second, the Risk Group obtained significantly poorer scores on the NEPSY than the Control Group. Third, on a cross-tabulation a very high rate of positive hits (93 %) was obtained as well as a very low rate of misses (7 %), indicating a very good sensitivity. However, there were a large percentage of false positives (63 %), indicating that specificity was not so good. Evidently, parents may report concerns related to the young child's development even when neuropsychological assessments do not indicate significant disorders. On the whole, the findings supported the validity of the FTF as a developmental screening instrument.

PURPOSE: There is controversy between the symptoms and signs of chondromalacia. Patellar chondromalacia has several clinical tests, whose reliability as a parameter of chondral damage is unclear. The purpose of this prospective study was to correlate the sensitivity, specificity, predictive values and accuracy of clinical patellar tests with the findings at arthroscopy. Type of Study: In this prospective study, 100 consecutive knees that were subjected to arthroscopy were examined. METHODS: Because of missing data, 85 of the 100 knees were included in the final analysis. There were 41 male and 44 female patients with an average age of 39 and 44 years, respectively. The clinical tests were the tracking test, the apprehension test, the patellar inhibition test, and the flexion test. These tests were compared with the arthroscopic findings of the patellar cartilage. The classification of Outerbridge was used for evaluation of the condition of the patellar cartilage. RESULTS: At arthroscopy, there were no patellar cartilage changes in 33 knees. Patellar chondromalacia was seen in 52 knees. Grade I changes were found in 9 knees, grade II in 21 knees, grade III in 17 knees, and grade IV in 5 knees. Among the 4 clinical tests, the sensitivity was best for the tracking test (56%). The flexion test had the greatest specificity (85%), but a low sensitivity (35%). None of the tests showed acceptable results in terms of both sensitivity and specificity. The predictive values and the accuracy of a test were low, too. CONCLUSIONS: The sensitivity and specificity, predictive values, and accuracy of a test were generally low, except perhaps the specificity of the flexion test. The current clinical tests seem to have little value as indicators of patellar chondral pathology.

The present study characterized the intensity-response functions of extracellular field responsiveness of different cortical/subcortical structures of the forebrain following the free-field presentation of tone stimuli, within a population of genetically audiogenic seizure (AGS)-prone KM-Wistar rats. The neural response properties of each case were compared to its propensity to exhibit AGSs during the continuous tone stimulation (15 kHz, 90 s at max.). The amplitudes or slope components of the evoked responses and their peak latencies showed significant positive (amplitude and slope) and negative (peak latency) Bolzmann's sigmoid relationships with the onset-latency of AGS. These relationships, with areal differences in the slopes of saturation functions, applied for the three different data sets recorded simultaneously from the stratum radiatum dendritic layer of the hippocampal CA1 area, primary auditory cortex layers II-IV, and frontal cortex surface. In addition, the similar type of functions between the evoked response variables and AGS onset latency held when all the areas were considered together. These data suggest that the neural responsiveness to acoustic stimulation of the primary sensory, multimodal and association cortices of the forebrain may altogether contribute to the seizure initiation by that modality in the genetically AGS-prone rats. It has been previously shown that there exist abundant and dispersed auditory projections from these forebrain areas to the brain stem and spinal cord, structures that are generally considered to be the key predisposing factors in the generation of AGS. Hence, the types of correlation found reflect the subject-specific stage of forebrain responsiveness, being either related or unrelated to genetic AGS-specific changes, and possibly its triggering impact upon the lower brain AGS network. Accordingly, the mere comparison of forebrain response measures of these AGS-prone animals with those of the AGS-resistant ones could not reveal the result presented.

ERp60 (also known as ERp61 or GRP58) is an isoform of protein disulfide isomerase (PDI) that has two thioredoxin-like domains a and a' in positions corresponding to those of domains a and a' in the PDI polypeptide and shows a significant amino acid sequence similarity to PDI in almost all parts. We report here that the human ERp60 gene is about 18 kb in size and consists of 13 exons. No distinct correlation was found between its exon-intron organization and the modular structure of the ERp60 polypeptide, nor were any similarities in exon-intron organization found between the human ERp60, PDI, and thioredoxin genes. The 5' flanking region of the ERp60 gene has no TATAA box or CCAAT motif but contains several potential binding sites for transcription factors. The highest levels of expression of the ERp60 mRNA were found by Northern blotting in the liver, placenta, lung, pancreas, and kidney, and the lowest in the heart, skeletal muscle, and brain. We also isolated an intronless ERp60 gene that probably represents a pseudogene. The ERp60 gene was mapped by fluorescence in situ hybridization to 15q15 and the processed gene to 1q21, so that neither was located on the same chromosome as the human PDI and thioredoxin genes.

Conclusion: The presence of the gallbladder at the onset of acute biliary pancreatitis is associated with increased severity of the disease. One possible explanation is that gallbladder contraction might induce bile reflux into the pancreatic duct during the transfer of a gallstone through the ampulla., Background: In clinical practice there is an impression that the presence of the gallbladder in patients with biliary pancreatitis may be associated with increased severity of the disease, compared to patients who have undergone cholecystectomy., Methods: To test this hypothesis, we studied 266 cases with biliary pancreatitis. Patients were divided into two groups: (A) those who had a gallbladder in situ at the onset of biliary pancreatitis (n = 234, 88%) and (B) those who had undergone previous cholecystectomy (n = 32, 12%)., Results: Pancreatitis was more severe in group A than in group B, according to Glasgow criteria (> or = 3 positive, 66/210 = 31% vs 4/29 = 14%, p = 0.04); development of complications (77/234 = 33% vs 4/32 = 13%, p = 0.01); and mortality (40/234 = 17% vs 1/32 = 3%, p = 0.03). Furthermore, serum C-reactive protein levels on admission were over 150 mg/L twice as often in group A as in group B.

In the current study, paclitaxel sensitivity of eight vulvar squamous cell carcinoma (SCC) cell lines was tested using a 96-well plate clonogenic assay. The chemosensitivity was expressed as IC50 corresponding to the drug concentration causing 50% inhibition in clonogenic survival. IC50 values were obtained from dose-response curves after fitting the data to the linear quadratic equation. The paclitaxel sensitivity of the eight vulvar SCC cell lines expressed as IC50 varied from 0.6 to 2.9 nM. The observed differences between the individual cell lines were surprisingly small, only 4.8-fold at the most. Paclitaxel sensitivity of vulvar SCC cell lines is of the same magnitude as the paclitaxel sensitivity of endometrial and ovarian carcinoma cell lines tested in our preliminary experiments with the same assay. We have previously studied the intrinsic radiosensitivity of these vulvar SCC cell lines. No correlation could be found between radiosensitivity and paclitaxel sensitivity. These results indicate that vulvar SCC is consistently sensitive to paclitaxel in vitro. The role of chemotherapy in the treatment of vulvar carcinoma has not been clarified yet. The efficacy of paclitaxel should be tested further in clinical trials.

Objective: To evaluate the occurrence, aetiology and outcome of acute pancreatitis (AP) in patients over 80 years of age, compared with those between 61 and 79 years old., Design: Retrospective analysis., Setting: University hospital, Finland., Subjects: Subgroups of 22 patients 80 years old or over and 139 patients 61-79 years old out of 1058 episodes of AP between 1982 and 1990., Main Outcome Measures: Mortality, morbidity, and hospital stay., Results: The percentage of patients aged 80 or more varied from 0 in 1982 to 3% in 1990; 13 (59%) were women compared with 47 (34%) in the 61-79 year old group (p = 0.03). AP in the older group was more likely to be necrotising 9/22 (41%) compared with 23/139 (17%), p = 0.02) and to have been caused by biliary disease 15/22 (68%) compared with 54/139 (39%), (p = 0.01). The overall mortality was 9/22 (41%) in the 80 years and over group and 24/139 (17%) in patients aged 61-79 years (p = 0.02). All 9 patients in their eighties with necrotising pancreatitis died. Neither the mode of treatment nor the Glasgow prognostic scoring had any relation to mortality in the older group., Conclusions: AP in patients of 80 or more is a serious disease with a high mortality irrespective of standard treatment.

Background: The antitubule agent paclitaxel causes a cell cycle blockage in the most radiosensitive part of the cell cycle, the G2/M phase. The possible radiosensitizing effect of paclitaxel was tested in four vulvar (UM-SCV-1A, UM-SCV-1B, UM-SCV-2, and UM-SCV-4) squamous cell carcinoma (SCC) cell lines., Methods: A 96-well plate clonogenic assay was performed with paclitaxel and radiation, both separately and concomitantly. Survival data were fitted to the linear quadratic model. The area under the curve, equivalent to the mean inactivation dose (D), was obtained by numerical integration. The effect of paclitaxel on radiosensitivity was measured as the AUC ratio (paclitaxel plus radiation: radiation alone). This ratio was compared with the surviving fraction (SFP) after paclitaxel alone., Results: Paclitaxel concentrations of 0.4 to 2.0 nanomolar (nM) caused 1 to 70% inhibition of clonogenic survival. The AUC values of the cell lines were 1.9 to 2.9 gray. A full additive effect was observed when paclitaxel and radiation were administered concurrently; however, a supra-additive effect never occurred. The type of paclitaxel radiation interaction was not affected by the concentration of the drug nor did the type of interaction vary between cell lines studied., Conclusions: Paclitaxel and radiation used concomitantly produced a clear additive effect at all concentrations and in all vulvar carcinoma cell lines tested. Although no supra-additive effect was observed, the additive effect already in nM concentrations could be beneficial in clinical use and, therefore, requires further investigation.

Tumour necrosis factor (TNF) is an early mediator of sepsis and multiple organ failure; increased concentrations in serum are also observed in acute pancreatitis. In the present study the predictive value of TNF and C-reactive protein (CRP) concentrations on admission were compared in order to differentiate complicated cases of acute pancreatitis from the mild course in 77 patients. Serum TNF concentration exceeded the detectable level only in seven of 77 patients (9 per cent), although complicated pancreatitis developed in 18 (23 per cent). The sensitivity and overall accuracy of TNF concentration in predicting severe disease were only 16 and 74 per cent respectively. The corresponding values for CRP (concentrations greater than 100 mg/l) were 84 and 74 per cent respectively. These data suggest that, in contrast with CRP, the early determination of peripheral blood TNF concentration is of no clinical value in assessing the severity of acute pancreatitis.

The prognosis of carcinomas arising from various sites in the head and neck varies even when the stage of the disease is taken into consideration, e.g. laryngeal carcinoma has a more favourable prognosis compared to oral-cavity malignancies. The purpose of this study was to evaluate intrinsic cellular radiosensitivity as one possible explanation for the observed differences in the survival rates of different anatomical groups. The radiation survival curves were determined for well characterized cell lines derived from laryngeal carcinoma (n = 14), pharyngeal carcinoma (n = 6), carcinoma of the oral cavity (n = 14) and the skin of the face (n = 3). The intrinsic radiosensitivity was expressed as area under the survival curve (AUC) values, and this cellular parameter was compared with clinical data and survival of the patients. The intrinsic radiosensitivity in the whole group varied between 1.0 Gy and 2.8 Gy with an average of 1.9 Gy. The mean AUC values for the laryngeal cell lines were 2.0 Gy +/- 0.2, for the oral cavity 1.8 +/- 0.3 Gy, for the pharynx 1.8 +/- 0.2 Gy and for cutaneous carcinoma 2.1 +/- 0.1 Gy. There was a slight difference between the groups of glottic and supraglottic cell lines (mean 1.8 +/- 0.2 Gy and 2.1 +/- 0.3 Gy, respectively), which is consistent with the differences in clinical curability of these cancers. Otherwise, the differences in cellular radiosensitivity of the carcinoma groups studied did not reach statistical significance. These results indicate that the intrinsic radiosensitivity of squamous-cell carcinoma (SCC) of the larynx does not significantly differ from that of SCC of other sites of the head and neck. Variations in the intrinsic radiosensitivity do not as such seem to explain the observed differences in radiocurability of SCC variously localized in the head and neck.

It has been shown recently that longterm but not short term heavy drinking of alcohol frequently results in increased serum activities of pancreatic enzymes suggesting subclinical pancreatic injury. Serum pancreatitis associated protein (PAP) is a novel protein, whose synthesis in the acinar cells and release into serum is specifically induced by acute pancreatic damage. This study was performed to further characterise the alcohol induced subclinical pancreatic injury by using serum PAP measurements. Three groups were studied: (1) control group (n = 25), (2) short term drinking group (n = 20), who consumed 2.0 g of ethanol per kg body weight during four hours, and (3) longterm drinking group (n = 32), who were admitted to withdrawal clinic after a median 30 months heavy drinking period. Serum PAP concentration was low in the control group (8 (5 to 12) micrograms/l, geometric mean (95% confidence intervals)). In the short term drinking group serum PAP was in the range of the control group values during 56 hours after drinking. Longterm drinking induced at least a 10-fold increase in serum PAP, the highest concentrations being seen on day 2 after drinking had ended (106 (61 to 184) micrograms/l). The patients did not develop abdominal symptoms, increased blood white cell count, or increased serum C reactive protein concentration. These results further support the suggestion that heavy longterm drinking often induces subclinical pancreatic damage, but not clinical pancreatitis.

UT-EC-2A was established from a patient with moderately differentiated Stage III endometrial adenocarcinoma with squamous metaplasia. UT-EC-2B was established from the same patient 17 months later from a metastasis in the left supraclavicular fossa. The origin of these cell lines was confirmed by DNA identity testing. Nude mice tumors produced by UT-EC-2A and UT-EC-2B cells recapitulated the histology of the original human tumors. Flow cytometric DNA contents of both primary and metastatic human tumors as well as corresponding nude mice tumors were diploid. The S-phase fractions of both cell lines were > or = 30%. The UT-EC-2A cell line was cytogenetically normal. The UT-EC-2B cell line had quite simple karyotype at low passage with an extra i(18p) and a deletion 21q, but at higher passages an additional three-way translocation 5;14;19 was observed. Radiosensitivity of the cell lines was tested with the 96-well plate clonogenic assay. The areas under the survival curves corresponding to the mean inactivation doses of UT-EC-2A and UT-EC-2B were 0.65 +/- 0.10 and 0.60 +/- 0.06 Gy, respectively. Measured survival at 2.0 Gy (SF2) was 0.042 for UT-EC-2A, 0.044 for UT-EC-2B, and 0.2 for skin fibroblasts. These cell lines are among the most radiosensitive human cancer cell lines described in the literature. Studying the characteristics of primary and metastatic cells derived from the same patient provides an opportunity to evaluate tumor progression.

Recently, high-dose short-term alcohol exposure has been observed not to induce acute pancreatic damage, as evaluated by serum pancreatic enzyme activities. In this study the effect of high-dose, long-term alcohol exposure on the pancreas was investigated in 32 consecutive alcoholics admitted to a unit to treat the problems of withdrawal after a long period of heavy drinking. None of the alcoholics complained of abdominal symptoms. The signs of clinical acute pancreatitis (pain, increased serum C-reactive protein concentration or blood white cell count) were not observed in any of the alcoholics. A significant increase in serum total amylase, pancreatic isoamylase and lipase activities developed by the second day after termination of alcohol intake. These enzyme activities remained significantly increased for one week after cessation of drinking. Seven alcoholics had signs of chronic pancreatitis at ultrasonography (pancreatic calcification, pseudocyst). These results suggest that heavy alcohol intake over the long term may frequently induce subclinical pancreatic injury.

We investigated the ability of various blood markers to detect an alcoholic cause of acute pancreatitis. Serum carbohydrate-deficient transferrin (CDT) was significantly correlated with reported 2 month and 7 day ethanol consumptions and was significantly higher in 42 patients with alcoholic acute pancreatitis and in 24 patients with possibly alcoholic acute pancreatitis than in 20 patients with non-alcoholic disease. At a cutoff over 17 U/L, the specificity of CDT was 100% and the sensitivity was 75% to detect an alcoholic cause of acute pancreatitis. The lipase/amylase ratio index, erythrocyte mean corpuscular volume, and gamma glutamyl transferase could not distinguish alcoholic from non-alcoholic acute pancreatitis.

Background: Does the amount of recently consumed alcohol correlate with the severity of acute alcoholic pancreatitis?, Methods: One hundred one consecutive episodes of acute pancreatitis (AP) were prospectively studied. Seventy-three were alcoholic AP episodes; 40 patients had their first alcoholic AP episode. A standard personal interview was used to determine the alcohol consumption during 2 months and during 1 week before AP. The severity of AP was evaluated according to the Ranson criteria, the serum C-reactive protein (CRP) concentration measured 24 to 48 hours after admission, the length of the hospital stay, the development of complications, and the mortality rate., Results: In the 40 patients having their first alcoholic AP episode, the reported 2-month alcohol consumption correlated significantly with the number of positive Ranson criteria (correlation coefficient r = 0.44, p < 0.01), serum CRP concentration (r = 0.51, p < 0.001), and the length of the hospital stay (r = 0.45, p < 0.01). Complications occurred in eight of 14 patients with 2-month alcohol consumption of more than 5000 gm as compared with one of 14 patients with consumption of less than 2000 gm (p < 0.05). In the same 40 patients the 1-week alcohol consumption correlated with the number of positive Ranson criteria (r = 0.40, p < 0.05) and serum CRP concentration (r = 0.37, p < 0.05). Of the 12 patients who had consumed more than 1000 gm alcohol during the last week before admission, two died and complications developed in six (50%), as compared with none (p < 0.05) and six (21%), respectively, of those who had consumed less than 1000 gm. No significant correlations were observed between the reported alcohol consumption and any of the severity parameters in the 33 patients with recurrent episodes of alcoholic AP., Conclusions: The amount of alcohol consumed may be an important determinant of the severity of the first alcoholic AP episode but not of recurrent alcoholic AP.

During 1982-90 we changed the surgical treatment of acute necrotizing pancreatitis twice. The operative strategy consisted of early formal resection (n = 13) during 1982-84, early lavational surgery (n = 15) during 1985-87 and late necrosectomy (n = 17) during 1988-90. The mortality rate decreased from 54% to 40% and to 24%, respectively. The pattern of complications changed from bleeding in resections to pseudocysts and fistulas in late necrosectomy. Sepsis and multiorgan failure were still main problems. These results led us to begin a prospective study of planned repeated debridement by open packing after delayed necrosectomy.

The incidence and mortality from pancreatitis in Finland between 1970 and 1989 were studied and compared with the alcohol consumption in the country and with the incidence of liver cirrhosis and gall stone disease. Hospital discharge data were obtained from the Finnish National Agency for Welfare and Health, the causes of deaths from the Finnish State Statistics, and annual alcohol consumption from the Finnish State Alcohol Company. There were 56,353 hospital treatment periods because of pancreatitis. The incidence of pancreatitis discharges increased from 46.6 to 73.4/100,000/year. In men it increased from 59.1 to 113.4, but in women it remained unchanged (mean 35.0). The incidence of pancreatitis discharges correlated with the alcohol consumption in Finland (r = 0.78, p = 0.0001). The incidence of pancreatitis discharges correlated in men, but not in women, with the incidence of liver cirrhosis (r = 0.81, p = 0.0001). In women, but not in men, the incidence of pancreatitis discharges correlated with the incidence of gall stone disease discharges (r = 0.77, p = 0.0001). The incidence of discharges due to haemorrhagic pancreatitis and pancreatic abscess doubled in men and remained unchanged in women. Pancreatitis death rate decreased from 5.9% (men 4.8%, women 7.0%) to 2.6% (men 2.4%, women 2.7%).

Short-term effects of a high dose of ethanol on the serum activities of pancreatic enzymes were studied in young healthy adults. There were 10 males and 10 females in the study group, and two males and three females in the control group. In the study group, ethanol (2 g/kg of body weight) was given during 4 h, resulting in the blood ethanol level of 1.6 +/- 0.3 g/l at 6 h. No significant changes in serum pancreatic enzyme activities were observed in the control group during the 56-h experiment. In the study group, none of the individuals demonstrated a marked increase (double from the baseline) in serum pancreatic enzyme activities. In the male study group, the serum total amylase activity increased slightly at 6 h, but the serum pancreatic isoamylase activity remained unchanged. These data suggest that, in nonalcoholic individuals, short-term exposure to a high dose of ethanol does not induce such injury in the pancreas that would appear as a leak of enzymes from the acinar cells into the circulation.

The clinical course of 72 septicemic episodes or focal severe bacterial infections was monitored by daily measurements of serum C-reactive protein (CRP) in 59 children beyond the neonatal period, 19 of whom were immunocompromised. CRP was determined quantitatively by an immunoturbidimetric method from a finger prick sample until either clinical recovery occurred and antimicrobial therapy was discontinued or until the death of the patient. The primarily elevated CRP levels (greater than or equal to 20 mg/l) usually increased about for a day but then decreased rapidly, provided the patient recovered uneventfully. If not, CRP remained at a high level or reincreased after transient decrease. Behaviour of CRP was not affected by the immunologic status of the patient. This property makes CRP especially useful in immunocompromised patients in whom other commonly used laboratory parameters may fail.

The value of quantitatively determined C-reactive protein (CRP), measured from a finger prick sample for rapid detection of septicemia, was examined in 76 blood culture-positive infections in 54 immunocompetent and 18 compromised children; 73 patients with systemic viral infections served as controls. Development of a positive CRP reaction was also studied in 40 cases of acute epiglottitis. Beyond the neonatal age, an increased CRP value (greater than or equal to 20 mg/L) was found in 60 of 64 (94%) children with a positive blood culture for bacteria or fungus. By contrast, CRP remained below this value in 56 of 73 (77%) with viral infections. The immunologic status did not influence the CRP response. However, time had a highly significant (p less than 0.001) effect on CRP; a history of 6 to 12 hours of illness was required before CRP increased above normal. We conclude that CRP is a sensitive and rapidly reacting index in bacteremic infections. However, because other factors than septicemia also increase CRP, we deem a negative CRP value most informative; if two determinations taken several hours apart are less than 20 mg/L, the patient is very unlikely to have invasive bacterial infection.

T-helper cells (ThC) play an important role in the induction of both cytotoxic T-cell responses and B-cell responses against the grafted organ. Furthermore, ThC alone are capable of causing graft rejection in T cell-deprived mice and rats. In view of these observations we found it important to analyse the frequency and functions of donor-specific ThC in the allograft and in the recipient lymphoid system during the course of acute renal allograft rejection. A limiting dilution assay was developed which, due to the absence of exogenous interleukin 2 (IL-2) and the low numbers of stimulator cells used, appears to be highly selective for the proliferation of specific ThC. Kidney transplants were performed from LBN (RT1n) to congenic Lewis (RT1l) strain differing in major histocompatibility complex (MHC) only. The inflammatory (white) cells were recovered from the graft, and blood and recipient spleen and the frequency of RT1n-responding ThC were determined at different times after transplantation. In the kidney graft itself, the frequency of ThC responding to RT1n MHC antigens was 1:3000 on day 2 and increased to 1:670-1320 at the peak of inflammation. In the spleen, the frequency increased from 1:1000 on day 0 to 1:200 on day 8, and remained high even after the graft was rejected. In the blood, the frequency stayed at the 1:400-1:800 level, and increased to 1:200 only after the graft had been completely destroyed. Individual ThC clones deriving from limited dilution assays of kidney and spleen cells were recovered and expanded with irradiated donor cells without IL-2 and finally with exogenous IL-2 only. All clones showed the T-helper (W3/25) phenotype, seven out of eight tested clones showed a specific anamnestic response to RT1n alloantigens and no response to RT1l or RT1a in a secondary MLC, 12 out of 12 clones produced IL-2 and 11 out of 11 clones produced gamma interferon upon re-stimulation with relevant allogeneic cells, and eight out of ten clones collaborated with syngeneic B cells for Ig synthesis, indicating that they were indeed derived from specific ThC and/or from their precursors. Taken together, the results demonstrate that specific ThC and/or their precursors represent only a very small minority in the graft-infiltrating inflammatory population. This makes it most unlikely that the ThC themselves are responsible for graft destruction; the results indicate rather that a major role of ThC in situ may be instruction of immunologically specific and nonspecific components of inflammation.