A meta-analysis of blood cytokine network alterations in psychiatric patients: comparisons between schizophrenia, bipolar disorder and depression.

Schizophrenia, bipolar disorder and major depressive disorder (MDD) have all been associated with aberrant blood cytokine levels; however, neither the pattern of cytokine alterations nor the impact of clinical status have been compared across disorders. We performed a meta-analysis of blood cytokines in acutely and chronically ill patients with these major psychiatric disorders. Articles were identified by searching the PubMed, PsycInfo and Web of Science, and the reference lists of these studies. Sixty-eight studies met the inclusion criteria (40 schizophrenia, 10 bipolar disorder and 18 MDD) for acutely ill patients. Forty-six studies met the inclusion criteria (18 schizophrenia, 16 bipolar disorder and 12 MDD) for chronically ill patients. Levels of two cytokines (interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)), one soluble cytokine receptor (sIL-2R), and one cytokine receptor antagonist (IL-1RA) were significantly increased in acutely ill patients with schizophrenia, bipolar mania and MDD compared with controls (P<0.01). Following treatment of the acute illness, IL-6 levels significantly decreased in both schizophrenia and MDD (P<0.01); sIL-2R levels increased in schizophrenia; and IL-1RA levels in bipolar mania decreased. In chronically ill patients, the levels of IL-6 were significantly increased in schizophrenia, euthymic (but not depressed) bipolar disorder and MDD compared with controls (P<0.01). The levels of IL-1ß and sIL-2R were significantly increased in both chronic schizophrenia and euthymic bipolar disorder. Overall, there were similarities in the pattern of cytokine alterations in schizophrenia, bipolar disorder and MDD during acute and chronic phases of illness, raising the possibility of common underlying pathways for immune dysfunction. Effects of treatment on cytokines were more robust for schizophrenia and MDD, but were more frequently studied than for acute mania. These findings have important implications for our understanding of the pathophysiology and treatment of major psychiatric disorders.

Peripheral cytokine and chemokine alterations in depression: a meta-analysis of 82 studies.

OBJECTIVE: To conduct a systematic review and meta-analysis of studies that measured cytokine and chemokine levels in individuals with major depressive disorder (MDD) compared to healthy controls (HCs). METHOD: The PubMed/MEDLINE, EMBASE, and PsycINFO databases were searched up until May 30, 2016. Effect sizes were estimated with random-effects models. RESULT: Eighty-two studies comprising 3212 participants with MDD and 2798 HCs met inclusion criteria. Peripheral levels of interleukin-6 (IL-6), tumor necrosis factor (TNF)-alpha, IL-10, the soluble IL-2 receptor, C-C chemokine ligand 2, IL-13, IL-18, IL-12, the IL-1 receptor antagonist, and the soluble TNF receptor 2 were elevated in patients with MDD compared to HCs, whereas interferon-gamma levels were lower in MDD (Hedge's g = -0.477, P = 0.043). Levels of IL-1ß, IL-2, IL-4, IL-8, the soluble IL-6 receptor (sIL-6R), IL-5, CCL-3, IL-17, and transforming growth factor-beta 1 were not significantly altered in individuals with MDD compared to HCs. Heterogeneity was large (I2 : 51.6-97.7%), and sources of heterogeneity were explored (e.g., age, smoking status, and body mass index). CONCLUSION: Our results further characterize a cytokine/chemokine profile associated with MDD. Future studies are warranted to further elucidate sources of heterogeneity, as well as biosignature cytokines secreted by other immune cells.

Balloon dilatation of the Eustachian tube: an evidence-based review of case series for those considering its use.

BACKGROUND: Eustachian tube dysfunction is common and implicated in persistent middle ear disease. Recent studies suggest the promise of balloon dilatation of the Eustachian tube to modify local anatomy and physiology, restoring normal function. OBJECTIVES: A literature review of the outcomes of balloon dilatation of the Eustachian tube was conducted, assessing four criteria (tympanometry, otoscopy findings, Valsalva and subjective symptoms). Outcomes were divided into short term (≤ 6 months) and long term (>6 months). Rates and severity of complications were documented. We also assessed cost, learning curve and requisite training. SEARCH STRATEGY: Medline via PubMed was consulted with the following search request: 'eustachian tube' OR 'auditory tube' AND 'balloon'. No restrictions were placed on study date, type or language. Non-clinical studies, published abstracts and very small studies (<10 procedures) were excluded. RESULTS: Our search yielded 24 results and six case series, five of which met the inclusion criteria. Balloon dilatation has been performed on 375 Eustachian tubes (235 patients) and demonstrates clear short-term (<6 month) benefits across all recorded outcome measures in a majority of cases. 69 of 89 (78%) tympanogram profiles recorded preoperatively as abnormal (Type B/C/open) resolved to type A profiles postoperatively. 40 of 46 (87%) otoscopy findings preoperatively reported as abnormal (tympanic membrane retraction, perforation or otitis media with effusion) normalised postoperatively. The ability to perform a consistently positive Valsalva manoeuvre improved from 15 of 139 (11%) to 89 of 139 (64%) cases following dilatation. The two largest studies reported on 210 and 100 procedures and described symptom improvement in 67% of cases at 2 months and 71% at 26.3 weeks, respectively. An overall complication rate of ≈ 3% was observed, and no major adverse events are reported (0%). Cost and learning curve of the procedure were both deemed to be acceptable. CONCLUSION: Balloon dilatation of the Eustachian tube appears to be safe, effective and affordable. Like many newly introduced techniques, the evidence remains limited to non-controlled case series, with heterogeneous data collection methods and lacking long-term outcomes. However, short-term data provides promising, consistent results based on objective measures, and when used selectively in patients refractive to maximal existing therapy, balloon dilatation presents a potentially significant advance.

Expression of genetically determined diabetes and insulitis in the nonobese diabetic (NOD) mouse at the level of bone marrow-derived cells. Transfer of diabetes and insulitis to nondiabetic (NOD X B10) F1 mice with bone marrow cells from NOD mice.

The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse is controlled by at least three recessive loci, including one linked to the MHC. To determine whether any of these genetic loci exert their effects via the immune system, radiation bone marrow chimeras were constructed in which (NOD X B10)F1-irradiated recipients were reconstituted with NOD bone marrow cells. Unmanipulated (NOD X B10)F1 mice, or irradiated F1 mice reconstituted with F1 or B10 bone marrow, did not display insulitis or diabetes. In contrast, insulitis was observed in a majority of the NOD----F1 chimeras and diabetes developed in 21% of the mice. These data demonstrate that expression of the diabetic phenotype in the NOD mouse is dependent on NOD-derived hematopoietic stem cells. Diabetogenic genes in the NOD mouse do not appear to function at the level of the insulin-producing beta cells since NOD----F1 chimeras not only developed insulitis and diabetes but also rejected beta cells within pancreas transplants from newborn B10 mice. These data suggest that the beta cells of the NOD mouse do not express a unique antigenic determinant that is the target of the autoimmune response.

Genetic control of diabetes and insulitis in the nonobese diabetic (NOD) mouse.

Genetic analysis of the development of diabetes and insulitis has been performed in the nonobese diabetic (NOD) mouse strain, a model of insulin-dependent (type I) diabetes mellitus. (NOD X C57BL/10)F1, F2, and (F1 X NOD) first-, second-, and third-backcross generations were studied. The data obtained were consistent with the hypothesis that diabetes is controlled by at least three functionally recessive diabetogenic genes, or gene complexes, one of which is linked to the MHC of the NOD. In contrast, pancreatic inflammation leading to insulitis was found to be controlled by a single incompletely dominant gene. One of the two diabetogenic loci that is not linked to the MHC appears to be essential for the development of severe insulitis. This diabetogenic gene may be identical to the gene that controls the initiation of the autoimmune response that progresses to insulitis. Although this gene appears to be functionally recessive in its control of diabetes, it is incompletely dominant in its control of insulitis. The MHC-linked diabetogenic gene, although not required for the development of insulitis, apparently influences the progression of the autoimmune response since NOD MHC homozygotes in the backcross generations displayed the highest incidence and most severe cases of insulitis. Interestingly, we have found two MHC heterozygous backcross females that have become diabetic, suggesting that either the MHC-linked diabetogenic gene is not strictly recessive or that a recombination event has occurred between the diabetogenic gene and the K or I-A regions of the MHC. The third diabetogenic locus appears to influence the progression of severe insulitis to overt diabetes. In animals homozygous at this locus, diabetes may result from a decreased ability to develop a protective suppressor response to the autoimmune process.

Autoimmune syndromes in major histocompatibility complex (MHC) congenic strains of nonobese diabetic (NOD) mice. The NOD MHC is dominant for insulitis and cyclophosphamide-induced diabetes.

The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse is controlled by multiple genes. At least one diabetogenic gene is linked to the major histocompatibility complex (MHC) of the NOD and is most likely represented by the two genes encoding the alpha and beta chains of the unique NOD class II molecule. Three other diabetogenic loci have recently been identified in the NOD mouse and are located on chromosomes 1, 3, and 11. In addition to the autoimmune diabetes which is caused by destruction of the insulin-producing beta cells in the pancreas, other manifestations of autoimmunity are seen in the NOD mouse. These include mononuclear cell inflammation of the submandibular and lacrimal glands, as well as the presence of circulating autoantibodies. To determine the effect of the non-MHC diabetogenic genes on the development of autoimmunity, we constructed the NOD.B10-H-2b (NOD.H-2b) strain, which possesses the non-MHC diabetogenic genes from the NOD mouse, but derives its MHC from the C57BL/10 (B10) strain. The NOD.H-2b strain does not develop insulitis, cyclophosphamide-induced diabetes, or spontaneous diabetes. It does, however, develop extensive lymphocytic infiltrates in the pancreas and the submandibular glands that are primarily composed of Thy 1.2+ T cells and B220+ B cells. In addition, autoantibodies are present in NOD.H-2b mice which recognize the "polar antigen" on the insulin-secreting rat tumor line RINm38. These observations demonstrate that the non-MHC genes in the NOD strain, in the absence of the NOD MHC, significantly contribute to the development of autoimmunity. The contribution of a single dose of the NOD MHC to autoimmunity was assessed with a (NOD x NOD.H-2b)F1 cross. Although only approximately 3% of F1 females developed spontaneous diabetes, approximately 50% of both female and male F1 mice developed insulitis, and 25% of females and 17% of males became diabetic after treatment with cyclophosphamide. These data demonstrate that the MHC-linked diabetogenic genes of the NOD mouse are dominant with decreasing levels of penetrance for the following phenotypes: insulitis greater than cyclophosphamide-induced diabetes greater than spontaneous diabetes.

Limitations in fields of vision for simulated young farm tractor operators.

Farm tractors account for the majority of deaths and injuries among youths working on North American farms. A vehicle operator's field of vision is an important operational aspect for safe driving. However, very little is known about visual limitations of young tractor operators compared to adult operators. The main purpose of this study was to quantify limitations in fields of vision of children with different anthropometry. The study was based on assessment of 42 farm tractors in popular use in the USA. The results showed that youth operators typically had diminished fields of vision compared to the average adult operator. The degree of visual limitation is greatest for objects at close distances and when objects are straight in front of the operator/tractor. This has serious implications in terms of risks for runovers, rollovers and collisions. Study findings may help illuminate the development of policies and guidelines in tractor-related jobs for children. STATEMENT OF RELEVANCE: This study provides an ergonomic approach for evaluation of children's visual limitations in tractor operations. This approach could be used in other related cases, where children are allowed to operate vehicles.

Normal fertilization occurs with eggs lacking the integrin alpha6beta1 and is CD9-dependent.

Previous results, based on inhibition of fertilization by an anti-alpha6 integrin mAb (GoH3), suggest that the alpha6beta1 integrin on mouse eggs functions as the receptor for sperm (Almeida, E.A., A.P. Huovila, A.E. Sutherland, L.E. Stephens, P.G. Calarco, L. M. Shaw, A.M. Mercurio, A. Sonnenberg, P. Primakoff, D.G. Myles, and J.M. White. 1995. Cell. 81:1095-1104). Because the egg surface tetraspanin CD9 is essential for gamete fusion (Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Suzuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324) and CD9 is known to associate with integrins, recent models of gamete fusion have posited that egg CD9 acts in association with alpha6beta1 in fusion (Chen, M.S., K.S. Tung, S.A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P.W. Kincade, J.C. Herr, and J.M. White. 1999. Proc. Natl. Acad. Sci. USA. 96:11830-11835; Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Su- zuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324). Using eggs from cultured ovaries of mice lacking the alpha6 integrin subunit, we found that the fertilization rate, fertilization index, and sperm binding were not impaired compared with wild-type or heterozygous controls. Furthermore, a reexamination of antibody inhibition, using an assay that better simulates in vivo fertilization conditions, revealed no inhibition of fusion by the GoH3 mAb. We also found that an anti-CD9 mAb completely blocks sperm fusion with either wild-type eggs or eggs lacking alpha6beta1. Based on these results, we conclude that the alpha6beta1 integrin is not essential for sperm-egg fusion, and we suggest a new model in which CD9 acts by itself, or interacts with egg protein(s) other than alpha6beta1, to function in sperm-egg fusion.

Video-assisted thoracoscopic surgery for ectopic mediastinal parathyroid adenoma.

Background: Primary hyperparathyroidism (PHPT), caused by an ectopic mediastinal parathyroid adenoma, is uncommon. In the past, when the adenoma was not accessible from the neck, median sternotomy was advocated for safe and successful parathyroidectomy. Video-assisted thoracoscopic surgical (VATS) parathyroidectomy represents a modern alternative approach to this problem. Methods: Information on patients undergoing VATS was obtained from a specific database, including clinical presentation, biochemistry, preoperative imaging, surgical approach and patient outcomes. A comprehensive literature review was undertaken to draw comparisons with other publications. Results: Over a 2-year period, nine patients underwent VATS parathyroidectomy for sporadic PHPT. Five patients had persistent PHPT following previous unsuccessful parathyroidectomy via cervicotomy, and four had had no previous parathyroid surgery. The median duration of surgery was 90 (range 60-160) min. Eight patients were cured biochemically, with no major complications. One patient required conversion to a median sternotomy for removal of a thymoma that had resulted in false-positive preoperative imaging. Conclusion: With appropriate preoperative imaging, multidisciplinary input and expertise, VATS parathyroidectomy is an effective, safe and well tolerated approach to ectopic mediastinal parathyroid adenoma.

Antecedentes: El hiperparatiroidismo primario (primary hyperparathyroidism, pHPT) causado por un adenoma paratiroideo ectópico mediastínico es infrecuente. Hace años, cuando un adenoma no era accesible por vía cervical se propugnaba una esternotomía media para efectuar una paratiroidectomía segura y con éxito. La paratiroidectomía por cirugía toracoscópica asistida por video (video­assisted thoracoscopic surgical, VATS) es una alternativa moderna para el abordaje de esta patología. Métodos: La información de los pacientes tratados con VATS se obtuvo de una base de datos específica, incluyendo presentación clínica, bioquímica, radiología preoperatoria, abordaje quirúrgico y resultados de los pacientes. Se efectuó una revisión extensa de la literatura para efectuar comparaciones con otras publicaciones. Resultados: Durante un periodo de 2 años, 9 pacientes fueron tratados mediante paratiroidectomía por VATS debido a un pHPT esporádico, de los cuales 5 presentaban pHPT persistente después del fracaso de una paratiroidectomía por cervicotomía, mientras que los 4 restantes no habían sido operados previamente de cirugía paratiroidea. El tiempo medio operatorio fue de 101 minutos (rango 60­160). Ocho pacientes se curaron bioquímicamente, sin ninguna complicación mayor. Un paciente precisó conversión a una esternotomía media para extirpar un timoma que había sido un falso positivo en la radiología preoperatoria. Conclusión: La paratiroidectomía por VATS es una intervención efectiva, segura y bien tolerada para la extirpación de un adenoma ectópico mediastínico, siempre y cuando se disponga de radiología preoperatoria adecuada, equipo multidisciplinar y experiencia.

In vitro folate deficiency induces deoxynucleotide pool imbalance, apoptosis, and mutagenesis in Chinese hamster ovary cells.

The genetic and epigenetic effects of nutritional folate deficiency were studied in two Chinese hamster ovary (CHO) cell lines. The CHO-AA8 cell line (hemizygous at the aprt locus) and CHO-UV5 (DNA repair-deficient mutant of AA8) were cultured in Ham's F-12 medium or in custom-prepared Ham's F-12 medium lacking folic acid, thymidine, and hypoxanthine. Cells cultured acutely in the folate deficient medium exhibited initial growth arrest, followed by massive cell death and DNA fragmentation into nucleosomal multimers characteristic of apoptosis. Although prolonged culture in the folate deficient medium was cytostatic and lethal to the majority cells, minor subpopulations in both cell lines failed to initiate cell death, exhibited phenotypic abnormalities, and adapted a selective growth advantage under marginal folate conditions. These "resistant" clones exhibited major alterations in deoxynucleotide pools associated with an increase in mutant frequency at the aprt locus as detected by resistance to cytotoxicity in 8-azaadenosine. The mutation frequency in the DNA repair-deficient CHO-UV5 cells was approximately 100-fold greater than that in the parental AA8 clones, underscoring the importance of DNA repair under conditions of folate deficiency and nucleotide pool imbalance. The enhanced mutation frequency in the DNA repair-competent folate-deficient CHO-AA8 cells suggests that DNA repair activity is less effective under folate-deficient conditions. These results add to the accumulating clinical and experimental evidence relating chronic folate deficiency to genomic instability and carcinogenesis.

Breaks in genomic DNA and within the p53 gene are associated with hypomethylation in livers of folate/methyl-deficient rats.

Male weanling Fischer 344 rats were fed either a semipurified diet deficient in the methyl donors methionine, choline, and folic acid or a supplemented control diet for a period of 9 weeks. At intervals of 2, 5, and 7 days, 3 weeks, and 9 weeks after initiation of the respective diets, the relative level of DNA strand breaks and the degree of cytosine methylation were quantified in high molecular weight DNA and also within the p53 gene in liver samples from these rats. Genome-wide strand break accumulation was associated with progressive genomic hypomethylation and increased DNA methyltransferase activity. With the use of quantitative PCR as a gene-specific DNA strand break assay, unique DNA strand breaks were detected in exon 5 but not in exons 6-8 of the p53 gene, and were accompanied by significant p53 gene hypomethylation. DNA hypomethylation has been shown to alter the conformation and stability of the chromatin structure, rendering affected regions more accessible to DNA-damaging agents. To determine whether methylation status alters the sensitivity of DNA to strand breakage, DNA in isolated nuclei was methylated in vitro and exposed to endogenous calcium/magnesium-dependent endonuclease activated under defined conditions. The incidence of enzyme-induced DNA strand breaks was decreased significantly with increased DNA methylation. In nuclei isolated from livers of methyl-deficient rats, the hypomethylated DNA was found to be more sensitive to enzyme- and oxidant-induced DNA strand break induction. Taken together, these results provide evidence that DNA strand breaks are induced in high molecular weight DNA and also within the p53 gene in liver tissue from methyl-deficient rats. The increased incidence of these strand breaks in DNA from methyl-deficient rats may be related to alterations in chromatin accessibility associated with DNA hypomethylation.

Pegylated-liposomal doxorubicin versus doxorubicin, bleomycin, and vincristine in the treatment of AIDS-related Kaposi's sarcoma: results of a randomized phase III clinical trial.

PURPOSE: Kaposi's sarcoma (KS), the most common neoplasm in patients with AIDS, is a significant clinical problem for which current therapies are frequently unsatisfactory. We conducted a randomized phase III clinical trial to compare the efficacy and toxicities of a new form of therapy, pegylated-liposomal doxorubicin, with standard combination chemotherapy in patients with advanced AIDS-related KS (AIDS-KS). PATIENTS AND METHODS: Two hundred fifty-eight patients with advanced AIDS-KS were randomly assigned to receive either pegylated-liposomal doxorubicin (20 mg/m2) or the combination of doxorubicin (20 mg/m2), bleomycin (10 mg/m2) and vincristine (1 mg) (ABV) every 14 days for six cycles. Standard response criteria, toxicity criteria, and predefined indicators of clinical benefit were examined to evaluate outcomes. RESULTS: Among 133 patients randomized to receive pegylated-liposomal doxorubicin, one achieved a complete clinical response and 60 achieved a partial response for an overall response rate of 45.9% (95% confidence interval [CI], 37% to 54%). Among 125 patients randomized to receive ABV, 31 achieved a partial response (24.8%; 95% confidence interval [CI], 17% to 32%). This difference was statistically significant (P < .001). In addition to objective responses, prospectively defined clinical benefits and toxicity outcomes also favored pegylated-liposomal doxorubicin. CONCLUSION: Pegylated-liposomal doxorubicin is more effective and less toxic than the standard combination chemotherapy regimen ABV for treatment of AIDS-KS.

Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice.

The nonobese diabetic (NOD) mouse, a model of human type I diabetes, develops insulitis beginning at 4-6 wk of age. By 30 wk of age, 72% of females and 39% of males develop spontaneous diabetes, apparently because of an overwhelming autoimmune response to the insulin-producing beta-cells within the islets. To identify the immune mechanism responsible for destruction of beta-cells in the NOD mouse, we developed an adoptive transfer protocol that induces diabetes in NOD mice at an age when spontaneous diabetes is rarely observed. Splenocytes from overtly diabetic NOD mice were unable to transfer diabetes to very young (less than or equal to 6 wk) irradiated NOD mice but effectively transferred diabetes to irradiated NOD mice greater than 6 wk of age. In such transfers, overt diabetes was induced within 12-22 days in greater than 95% (79/82) of the recipients. Thus, transfer of splenocytes to young mice induces them to become diabetic at a higher frequency and at a younger age than their untreated littermates. Equally successful transfers with as few as 5 X 10(6) spleen cells have been performed in male and female NOD mice, even though males display a lower spontaneous incidence of diabetes than females. Splenocytes obtained from diabetic mice maintained on insulin for up to 2 mo also transferred diabetes. Because NOD mice display increasing levels of insulitis with age, spleen cells obtained from nondiabetic NOD mice of different ages were tested for their ability to transfer diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

A simplified HPLC method for simultaneously quantifying ribonucleotides and deoxyribonucleotides in cell extracts or frozen tissues.

Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle progression. A simplified procedure for the separation of both ribonucleoside triphosphates (NTP) and deoxyribonucleoside triphosphates (dNTP) is presented which utilizes reversed phase high-performance liquid chromatography coupled with diode array detection. The simultaneous resolution of NTP and dNTP peaks within the same cell extract effectively eliminates the need for post-extraction steps such as periodate oxidation and/or boronate affinity chromatography previously used to degrade or isolate co-eluting NTP from dNTP. The resolution of two nucleotides, dGTP and ADP, was found empirically to vary with the efficiency of the C18 column. High efficiency columns (> 90,000 plates/m) provided good separation; however, less efficient columns resulted in co-elution of dGTP and ADP. These co-eluting nucleotides can be accurately quantified, if necessary, using diode array technology and a mathematical expression which incorporates molar peak coefficients and peak areas obtained by monitoring at dual wave-lengths. Tissue samples or single cell suspensions were extracted with trichloroacetic acid and the neutralized extract was injected directly into the column without prior lyophilization. The per cent recovery of standards was > or = 99% and replicate extractions within or between samples were highly reproducible (SD < 5%). The single step method described minimizes potential losses associated with post-extraction manipulation and provides the capability to examine alterations in nucleotide precursor-product metabolism under various physiological and pharmacological conditions.

Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat.

Chronic dietary methyl deficiency in F344 rats was used as an in vivo mammalian model in which to evaluate the gene-specific alterations in DNA methylation patterns during multistage hepatocarcinogenesis. Using bisulfite mapping, the site-specific methylation profile within exons 6-7 of the 53 gene was determined in control liver, preneoplastic nodules (after 36 weeks of folate/methyl deficiency) and in hepatocellular carcinoma (after 54 weeks of deficiency). A progressive loss of methyl groups was observed at most CpG sites on both coding and non-coding strands during the first 36 weeks of folate/methyl deficiency, with the greatest loss occurring on the coding strand. When the same sequence was evaluated in tumor DNA after 54 weeks of deficiency, the majority of cytosines were unexpectedly found to have become remethylated. CpG sites that had previously lost methyl groups on both strands during preneoplasia as well as CpG sites that had been constitutively non-methylated, had undergone de novo methylation in tumor DNA. Maintenance methyltransferase and de novo methyltransferase activity in nuclear extracts were assessed using hemimethylated and non-methylated DNA substrates, respectively. In tumor, de novo methyltransferase capacity was increased approximately 4-fold relative to control or preneoplastic liver and associated with a relative increase in both p53 and genome-wide methylation density. In the preneoplastic nodules, the level p53 mRNA was increased and associated with hypomethylation in the coding region of the gene, whereas in tumor tissue, p53 mRNA was decreased and associated with relative hypermethylation. Taken together, these results provide additional insights into the dysregulation and instability in DNA methylation that accompanies the transition to tumor.

Uracil misincorporation, DNA strand breaks, and gene amplification are associated with tumorigenic cell transformation in folate deficient/repleted Chinese hamster ovary cells.

Clinical and experimental evidence has linked nutritional folic acid status to both anti- and procarcinogenic activity. Folate supplementation of normal cells appears to have a protective effect; however, folate supplementation of initiated cells may promote neoplastic progression. Given these considerations, the present series of experiments examines alterations in DNA metabolism and cumulative DNA lesions using an in vitro model of folate deprivation and repletion. DNA repair-deficient CHO-UV5 cells were cultured in Ham's F-12 medium or in custom-prepared Ham's F-12 medium lacking in folic acid, thymidine and hypoxanthine for a period of 18 days without cell passage. The results indicated that progressive folate and nucleotide depletion leads to a significant increase in the ratio of dUTP/dTTP and to the misincorporation of uracil into DNA. These alterations were accompanied by growth inhibition, DNA strand breaks, abasic sites and phenotypic abnormalities. After 14 days in culture, there was significant increase in gene amplification potential in the chronically folate-deficient cells, but no significant increase in anchorage-independent growth or in neoplastic transformation. Acute folate repletion of the deficient cells was used as a proliferative stimulus under conditions of dNTP pool imbalance and multiple lesions in DNA. A further increase in gene amplification was accompanied by anchorage-independent growth and neoplastic cell transformation as evidenced by aggressive tumor growth in Balb/c nu/nu mice. Using a sensitive in vitro model system, these results emphasize the essentiality of folic acid for de novo nucleotide synthesis and the integrity of the DNA. However, the in vivo relevance, especially in terms of tumorigenic potential, is not clear.

The essentiality of folate for the maintenance of deoxynucleotide precursor pools, DNA synthesis, and cell cycle progression in PHA-stimulated lymphocytes.

The fidelity and progression of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) precursors for the polymerases involved in DNA replication and repair. Because folate-derived one-carbon groups are essential for the de novo synthesis of both purines and pyrimidines, the purpose of this study was to determine the effect of folate deprivation on deoxynucleotide pool levels and cell cycle progression. Primary cultures of phytohemagglutin (PHA)-stimulated splenocytes were used as the cellular model. T-cells and macrophages were purified from spleen cell suspensions obtained from F344 rats and recombined in culture. The cells were harvested after a 66-hr incubation with PHA and analyzed for nucleotide levels by reverse-phase HPLC with diode array detection. The proportion of cells in the different phases of the cell cycle was determined by bivariate flow cytometric measurement of bromodeoxyuridine (BrdU) incorporation and DNA content (propidium iodide staining). PHA-stimulated T-cells cultured in medium lacking folate and methionine manifested significant decreases in the deoxynucleotides dCTP, dTMP, dGTP, and dATP relative to cells cultured in complete medium. The reduction in dNTP pools was associated with a decrease in the corresponding ribonucleotide pools. Flow cytometric analysis revealed a 2-fold increase in S and G2/mitosis (G2/M) DNA content in PHA-stimulated cells cultured in the medium lacking folate and methionine, which suggests a delay in cell cycle progression. These alterations in DNA content were accompanied by a 5-fold decrease in BrdU incorporation relative to PHA-stimulated cells cultured in complete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cellular response to silicone implants in rats: implications for foreign-body carcinogenesis.

Foreign-body (FB) carcinogenesis is a classic model of multistage tumour development in rodents. Previous studies have demonstrated that the physical characteristics of the implant, and not the chemical composition, are the critical determinants of tumour development. The recent controversy over silicone breast implants has raised questions regarding the potential carcinogenicity of lifetime tissue exposure to silicone products. The present study was designed to determine whether the inflammatory and fibrotic reactions associated with silicone implants are due to a non-specific foreign-body reaction or whether these responses reflect the unique chemical composition of silicone. F344 rats were implanted subcutaneously with one of three biomaterials: silicone elastomer (Group 1); impermeable cellulose acetate filters (Group 2, positive control); or porous cellulose acetate filters (Group 3, negative control). The silicone and cellulose implants of Groups 1 and 2 have been previously shown to induce fibrosarcomas in rodents, whereas the porous cellulose acetate implants of Group 3 have been shown to be non-carcinogenic. One week and two months after implantation, the pericapsular tissues were evaluated using histopathological and in situ immunohistochemical analyses. Endpoints included expression of leucocyte antigens CD4 (T helper/inducer), CD8 (T suppressor/cytotoxic) and CD11 b/c (macrophage), proliferating cell nuclear antigen (PCNA) as an indicator of proliferation, and in situ end-labelling (ISEL) of 3'OH DNA strand breaks as an indicator of DNA damage and apoptosis. The results indicated that the acute and chronic cellular responses to silicone (Group 1) were not different from impermeable cellulose filters (Group 2) of identical size and shape, suggesting that these responses were not unique to silicone. The inflammatory response to the carcinogenic cellulose and silicone implants (Groups 1 and 2) was attenuated and associated with the formation of a thick fibrotic capsule. In contrast, the porous cellulose filters (Group 3) induced a markedly different cellular response in which the inflammatory reaction was more extensive, prolonged and associated with minimal fibrosis. Within the fibrotic capsule surrounding the tumorigenic implants, but not the non-tumorigenic implants, cell proliferation and apoptotic cell death were increased and associated with persistent DNA strand breaks. Taken together, the results suggest that the micrometre-scale surface morphology of the implant determines the nature of the subsequent cellular response which may predispose to tumour development. Further, these studies serve to emphasize the critical importance of appropriate physical controls in studies designed to evaluate carcinogenic or autoimmune manifestations associated with silicone implants in order to rule out the contribution of the chronic foreign-body reaction.

Kaposi sarcoma of the conjunctiva and eyelids associated with the acquired immunodeficiency syndrome.

Three studies were performed to assess more accurately the prevalence, natural history, and appropriate treatment of acquired immunodeficiency syndrome (AIDS)-related Kaposi sarcoma involving ocular structures. The first study was a prospective examination of 100 male homosexuals with AIDS-related Kaposi sarcoma for signs of ophthalmic involvement. Of the 20 patients who had ophthalmic lesions, 16 had eyelid lesions and seven had conjunctival lesions. In four patients, the ophthalmic lesion was the first, and initially the only, clinically identified manifestation of Kaposi sarcoma. The second study was a retrospective review of all patients with ophthalmic Kaposi sarcoma examined at one institution over a six-year period to determine its natural history and response to therapy. Most lesions were slowly progressive and responded to systemic drug therapy. Six patients were successfully treated with radiation therapy to prevent complications. The third study was a retrospective review of all patients with AIDS-related ophthalmic Kaposi sarcoma treated with local irradiation by one radiation oncologist. Each of 12 patients showed a response to treatment, and ten had a complete resolution of lesions, but recurrences were common. Side effects included skin erythema in six patients and hair loss in one patient. For local treatment of ophthalmic Kaposi sarcoma, irradiation appears to be safe and effective for palliative therapy.

Metastatic pancreatic adenocarcinoma to the heart diagnosed antemortem.

Pancreatic tumors frequently metastasize widely, though it is rare to diagnose pancreatic cardiac metastases in the antemortem state. We report an unusual case of metastatic pancreatic adenocarcinoma to the right atrium. Transesophageal echocardiography showed that the tumor was attached to the superior aspect of the right atrium, prolapsing through and obstructing the tricuspid valve in diastole and retracting back into the right atrium during systole. The tumor was excised, and histologic examination confirmed the presence of moderately differentiated adenocarcinoma with a papillary architectural pattern and with desmoplastic stroma, features comparable to the original primary pancreatic neoplasm.