Band 28, Heft 3-4

Proteins are one of the preferential targets of the photosensitized damaging effects of ultraviolet (UV) radiation on biological system. Pterins belong to a family of heterocyclic compounds, which are widespread in living systems and participate in relevant biological functions. In pathological conditions, such as vitiligo, oxidized pterins accumulate in the white skin patches of patients suffering this depigmentation disorder. It is known that pterins are able to photosensitize damage in nucleotides and DNA by type I (electron transfer) and type II (singlet oxygen) mechanisms. Recently, it has been demonstrated that proteins and its components may also be damaged when solutions containing both proteins and pterin are exposed to UV-A radiation. Therefore, given the biological and medical relevance of the photosensitizing properties of these molecules, we present in this article an overview of the capability of different pterin derivatives to photoinduce damage in proteins present in the skin, focusing our attention on the chemical modifications of tyrosine and tryptophan residues.

Clinical practice and experimental studies have shown the necessity of sufficient quantities of folic acid intake for normal embryogenesis and fetal development in the prevention of neural tube defects (NTDs) and neurological malformations. So, women of childbearing age must be sure to have an adequate folate intake periconceptionally, prior to and during pregnancy. Folic acid fortification of all enriched cereal grain product flour has been implemented in many countries. Thus, hundreds of thousands of people have been exposed to an increased intake of folic acid. Folate plays an essential role in the biosynthesis of methionine. Methionine is the principal aminopropyl donor required for polyamine biosynthesis, which is up-regulated in actively growing cells, including cancer cells. Folates are important in RNA and DNA synthesis, DNA stability and integrity. Clinical and epidemiological evidence links folate deficiency to DNA damage and cancer. On the other hand, long-term folate oversupplementation leads to adverse toxic effects, resulting in the appearance of malignancy. Considering the relationship of polyamines and rapidly proliferating tissues (especially cancers), there is a need for better investigation of the relationship between the ingestion of high amounts of folic acid in food supplementation and polyamine metabolism, related to malignant processes in the human body.

In our individual and collaborative studies, we have played a part in pioneering investigations on the usefulness of biomarkers – red blood cell distribution width (RDW) and neopterin. This mini review includes historical data on the topic and is related to the first contributions in this field, as well as to the possibilities for further improvement and simultaneous application of RDW and neopterin measurements in the prevention, prognosis and treatment of a great number of socially important disease conditions (arterial, cardiovascular, brain vascular, peripheral artery diseases, inflammations, autoimmune states, cancers and leukemias, addictions, etc.). When comparing the results obtained with the immunobiochemical biomarker neopterin with RDW, they are reported to be very similar as independent predictors of the same pathological states in the human body although their biomedical origins are very different. Both the parameters were until now successfully, but only separately used in medical practice. The combined use of these two biomarkers can shed some more light on their interrelationships and provide some clues as to how the interaction between immune system activation and red blood cells biology are intertwined.

As early as 1974, reports have confirmed the anticancer activity of pterin deaminase isolated from fungi. The enzyme has also been reported in bacteria, fungi and slime mold genera, but the enzyme characterization was effetely done. The present study attempted to purify and characterize pterin deaminase enzyme from Saccharomyces cerevisiae NCIM 3458. The protein was extracted from the extracellular extract by using the ethanol precipitation method. Partial purification of pterin deaminase enzyme was achieved by ion exchange chromatography (Hi-Trap QFF) by fast protein liquid chromatography (AKTA purifier). The molecular weight of the protein was apparently determined by SDS-PAGE, and the presence of pterin deaminase was confirmed by activity staining. The purified enzyme was further biochemically characterized. Molecular docking studies showed higher binding affinity towards folic acid interaction. The structural characterization of this protein may open the windows for new drug targets for cancer therapy.

We have developed a fluorescence assay system to monitor the protein levels of human phenylalanine hydroxylase (hPAH). Wild-type (WT) and three mutant hPAHs (I65T, L255V, and S349L) were expressed as green fluorescent protein (GFP)-tagged forms in a PAH knockout mutant ( pah − ) of Dictyostelium discoideum Ax2. The fluorescence-activated cell sorting (FACS) analysis showed that the GFP positive cells were the most frequent in WT but were rare in pah − , demonstrating the successful expression of GFP-tagged hPAHs in Dictyostelium . The fluorescence levels of mutants relative to WT were higher than expected from the protein amounts determined from the non-tagged forms, probably due to the presence of the N-terminal GFP. However, treatment of the cells with cumene hydroperoxide, which is known to accelerate protein degradation, decreased fluorescence levels, suggesting that protein stability changes in individual mutations can be monitored by FACS analysis. For an evaluation study, a putative pharmacological chaperone effect of yeast extract on S349L was examined by Western blot and FACS analysis. Both the protein amount and the fluorescence levels were increased by yeast extract, supporting that the FACS analysis could replace the time- and labor-consuming procedures such as the Western blot and cell culture. The fluorescence-based cell assay system may be valuable for the high-throughput screening of pharmacological chaperones for phenylketonuria mutations.

Determining the accurate age of malaria vectors is crucial to measure the risk of malaria transmission. A group of fluorescent chemicals derived from a pyrimidine-pyrazine ring structure known as pteridines from the head, thorax and whole body of adult female Anopheles stephensi were identified and evaluated as a tool for chronological and physiological age determination of malaria vectors. The female mosquitoes were collected from an insectary colony at an interval of every 5 days, up to 30 days, and the pteridines of head, thorax and whole body were detected fluorometrically by high-pressure liquid chromatography (HPLC) using excitation and emission wavelengths of 365 and 455 nm, respectively. In addition, alteration of the pteridines compounds was compared between blood and sugar fed mosquito groups. Although four pteridines including pterin-6-carboxylic acid, biopterin, xanthopterin and isoxanthopterin were detected, some of them were absent in the head or thorax of mosquitoes. Levels of all four pteridines were similarly decreased in a linear manner throughout 30 days. No significant difference in alteration of pteridine compounds was observed between the two groups of blood or sugar fed mosquitoes. This result indicates that diet has a little effect on pteridines alteration. Age determination based on pteridines, as an age-grading technique, could be used for field collected mosquitoes, which have either sugar or blood meal. In addition, analyzing total pteridine fluorescence from only whole body could be a convenient method to estimate the age.

Seasonal changes in non-human animals and seasonal affective disorder (SAD) in humans are associated with immune activation in winter relative to summer. We intended to measure seasonal variation in neopterin, a marker of cellular immunity, and its interactions with gender and seasonality of mood. We studied 320 Amish from Lancaster, PA, USA (men=128; 40%) with an average age [Standard deviation (SD)] of 56.7 (13.9) years. Blood neopterin level was measured with enzyme-linked immunosorbent assay (ELISA). Seasonality was measured with Seasonal Pattern Assessment Questionnaire (SPAQ). Statistical analysis included analysis of covariance (ANCOVAs) and multivariate linear regression. We also investigated interactions of seasonal differences in neopterin with gender, seasonality scores and estimation of SAD diagnosis. We found a significantly higher neopterin level in winter than in summer (p=0.006). There were no significant gender or seasonality interactions. Our study confirmed the hypothesized higher neopterin level in winter. A cross sectional design was our major limitation. If this finding will be replicated by longitudinal studies in multiple groups, neopterin could be used to monitor immune status across seasons in demographically diverse samples, even if heterogeneous in gender distribution, and degree of seasonality of mood.

Neopterin is a novel predictor for coronary events especially in diabetic patients and also an indicator for the effectiveness of the periodontal treatment. In this study, we assessed whether salivary neopterin can be used as a potential biomarker in evaluating the risk of cardiovascular disease in type 2 diabetic patients with chronic periodontitis. Forty subjects between 25 and 75 years of age and who matched the criteria were selected and divided into four groups. Their periodontal status was evaluated. Stimulated whole saliva and blood were collected for analysis of salivary neopterin and fibrinogen and HbA 1c levels, respectively. Nonsurgical periodontal therapy was carried out. Patients were recalled after 3 months, and the same procedure was repeated. A reduction in all the parameters was seen after treatment in all the four groups. Salivary neopterin levels showed significant difference (p<0.001) in the values between the study groups and the control group before treatment. After 3 months of treatment, salivary neopterin levels showed a statistical significant reduction (p<0.001) in all the study groups. Neopterin could serve as an effective tool to assess the inflammatory process related to periodontitis and diabetes mellitus and also predict future cardiovascular events in diabetic patients.

Toxoplasma gondii ( T. gondii ) IgG seropositivity and serointensity have been previously associated with suicidal self-directed violence (SSDV). Although associations with unipolar depression have also been investigated, the results have been inconsistent, possibly as a consequence of high heterogeneity. We have now studied this association in a more homogeneous population, [that is (i.e.) Old Order Amish (OOA)] with previously reported high T. gondii seroprevalence. In 306 OOA with a mean age of 46.1±16.7 years, including 191 (62.4%) women in the Amish Wellness Study, we obtained both T. gondii IgG titers (by enzyme-linked immunosorbent assay [ELISA]), and depression screening questionnaires (Patient Health Questionnaire [PHQ-9] [n=280] and PHQ-2 [n=26]). Associations between T. gondii IgG and dysphoria/hopelessness and anhedonia scores on depression screening questionnaires were analyzed using multivariable linear methods with adjustment for age and sex. Serointensity was associated with both current dysphoria/hopelessness (p=0.045) and current combined anhedonia and dysphoria/hopelessness (p=0.043), while associations with simple anhedonia and past/lifelong (rather than current) phenotypes were not significant. These results indicate the need for larger longitudinal studies to corroborate the association between dysphoria/hopelessness and T. gondii IgG-titers. Current hopelessness is a known risk factor for SSDV which responds particularly well to cognitive behavioral therapy, and may be a focused treatment target for T. gondii -positive individuals at high-risk for SSDV.

Toxoplasma gondii ( T. gondii ) infects central nervous tissue and is kept in relative dormancy by a healthy immune system. Sleep disturbances have been found to precipitate mental illness, suicidal behavior and car accidents, which have been previously linked to T. gondii as well. We speculated that if sleep disruption, particularly insomnia, would mediate, at least partly, the link between T. gondii infection and related behavioral dysregulation, then we would be able to identify significant associations between sleep disruption and T. gondii . The mechanisms for such an association may involve dopamine (DA) production by T. gondii , or collateral effects of immune activation necessary to keep T. gondii in check. Sleep questionnaires from 2031 Old Order Amish were analyzed in relationship to T. gondii -IgG antibodies measured by enzyme-linked immunosorbent assay (ELISA). Toxoplasma gondii seropositivity and serointensity were not associated with any of the sleep latency variables or Epworth Sleepiness Scale (ESS). A secondary analysis identified, after adjustment for age group, a statistical trend toward shorter sleep duration in seropositive men (p=0.07). In conclusion, it is unlikely that sleep disruption mediates links between T. gondii and mental illness or behavioral dysregulation. Trending gender differences in associations between T. gondii and shorter sleep need further investigation.

The aim of the present pilot study was to investigate the concentrations of neopterin, kynurenine and tryptophan in wound secretion in patients undergoing surgery for breast cancer or malignant melanoma. Twenty-two patients, 16 females and 6 males, undergoing surgery for breast cancer (n=15) or malignant melanoma (n=7) were evaluated. Neopterin, kynurenine and tryptophan were determined using a high-performance liquid chromatography method. When the concentrations in wound secretions from the primary breast tumor and the axilla were compared, the neopterin/tryptophan ratio was significantly higher in the tumor wound secretions (0.92±0.41 vs. 0.61±0.14 mmol/mol; p=0.049), but no significant differences were observed in neopterin (49.2±28.6 vs. 31.5±11.1 nmol/L), tryptophan (52.9±13.0 vs. 51.2±13.3 μmol/L) and kynurenine concentrations (5.97±7.49 vs. 5.34±6.25 μmol/L) and kynurenine/tryptophan ratio (108.1±107.7 vs. 103.5±106.7 mmol/mol). No marked differences were noted in neopterin, tryptophan and kynurenine concentrations and kynurenine/tryptophan and neopterin/tryptophan ratios in sequential samples from the axilla of breast cancer patients obtained on days 1 and 2. In conclusion, present data demonstrate that the measurement of neopterin, kynurenine and tryptophan can be used to monitor local immune response after cancer surgery.

In the present study, we determined complex indices of inflammatory activity and compared the performance of these indices as prognostic biomarkers in a cohort of breast cancer patients. All proposed composite biomarkers could be evaluated in 418 out of 474 patients in the cohort with complete data on peripheral blood cell count, urinary neopterin, albumin and C-reactive protein. Neutrophil-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, platelet-to-lymphocyte ratio, systemic inflammatory index, Glasgow prognostic index, modified Glasgow prognostic index, prognostic nutritional index and C-reactive protein/albumin ratio were calculated and further complex indices were proposed. Although a number of the investigated indices were significantly associated with survival in the univariate analysis, only age and stage, but none of the laboratory biomarkers or composite biomarkers, were significant predictors of survival in the whole group in the multivariate analysis. In patients evaluated before the start of the treatment, age, stage and urinary neopterin were significant predictors of survival. These results underscore the importance of neopterin as a prognostic biomarker in breast cancer.

The aim of the present study was to investigate the association of peripheral-blood cell count (PBC)-derived ratios, other biomarkers of inflammation and biomarkers of tumor growth with outcome in a cohort of patients presenting for the next line of therapy after the failure of prior systemic treatment. The data of 51 patients with advanced/metastatic colorectal carcinoma treated with cetuximab in the second or higher line of therapy were retrospectively analyzed. The median duration of cetuximab therapy and the median survival were 5.1 and 12.1 months, respectively. C-reactive protein (CRP), but not urinary neopterin correlated significantly with PBC-derived ratios. Both CRP and urinary neopterin correlated positively with carcinoembryonic antigen (CEA) concentrations and biomarkers of liver dysfunction. Although a number of parameters predicted overall survival in univariate analysis, only hemoglobin, CEA change and serum bilirubin were independent predictors of survival. In conclusion, in patients with metastatic colorectal carcinoma and predominantly liver metastases, the outcome of therapy in the advanced line setting was associated with initial hemoglobin level, a decrease of CEA concentration and initial presence of liver dysfunction. Urinary neopterin did not correlate with PBC-derived ratios, in contrast to CRP, but both urinary neopterin and serum CRP concentrations correlated with laboratory parameters of liver dysfunction.

We report a patient who presented with synchronous second primary human epidermal growth factor receptor (HER)-2-positive breast cancer and rectal cancer that both required simultaneous neoadjuvant therapy. A modified regimen combining anti-HER-2 monoclonal antibody trastuzumab with chemotherapy and external beam radiation was selected. An organ-preserving surgical procedure was possible both in the breast and the rectum. Citrulline decreased rapidly after the start of the treatment, and then gradually returned to pre-treatment levels after the completion of chemoradiation. Urinary neopterin concentrations exhibited a fluctuating course. Both serum neopterin and C-reactive protein concentrations were more or less stable during the initial administration of trastuzumab, paclitaxel and carboplatin and then increased steeply during chemoradiation and subsequently declined to pre-treatment levels during the weekly trastuzumab administration. Changes were observed in the serum retinol concentrations. A decline in lymphocyte counts was accompanied by marked changes in peripheral blood cell count-derived ratios. The present case report demonstrates a successful combination of two neoadjuvant regimens in a patient with two synchronous different second primary tumors. Data from this case also illustrate the use of biomarkers for monitoring of intensive therapeutic regimens in medical and radiation oncology.