Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the small intestine and colon caused by a dysregulated immune response to host intestinal microbiota in genetically susceptible subjects. A number of fermented dairy products contain lactic acid bacteria (LAB) and bifidobacteria, some of which have been characterized as probiotics that can modify the gut microbiota and may be beneficial for the treatment and the prevention of IBD. The objective of this review was to carry out a systematic search of LAB and bifidobacteria probiotics and IBD, using the PubMed and Scopus databases, defined by a specific equation using MeSH terms and limited to human clinical trials. The use of probiotics and/or synbiotics has positive effects in the treatment and maintenance of UC, whereas in CD clear effectiveness has only been shown for synbiotics. Furthermore, in other associated IBD pathologies, such as pouchitis and cholangitis, LAB and bifidobacteria probiotics can provide a benefit through the improvement of clinical symptoms. However, more studies are needed to understand their mechanisms of action and in this way to understand the effect of probiotics prior to their use as coadjuvants in the therapy and prevention of IBD conditions. Inflammatory bowel disease (IBD) can be defined as a disease of disrupted physiology, microbiology, immunology, and genetics . IBD maiMicroorganisms in the human gut act in symbiosis to modulate different functions, such as the stimulation-regulation of epithelial innate immunity, the competitive exclusion of pathogens, and the production of important metabolites ) –5.Traditional fermented products, breast milk, gastrointestinal tract content, and the feces of human subjects are the primary sources of LAB and bifidobacteria . LAB andProbiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” according to the consensus of a multinational expert group of scientists convened in 2001 by the Food and Agriculture Organization of the United Nations (FAO) . The terThe main aim of this work was to review the scientific evidence on the role of LAB and bifidobacteria, which are commonly used as probiotics, mainly in the prevention and treatment of IBD and other related IBD. In addition, we provide potential mechanisms of action of LAB and bifidobacteria in those conditions.In this paper, we performed a systematic review of the role of fermented dairy products and LAB and bifidobacteria probiotics in the prevention and treatment of IBD. PubMed and Scopus were searched for human randomized clinical trials articles that were published between 1990 and June 2014 in English using the MeSH terms “dairy products” and “probiotics” combined with “inflammatory bowel disease,” “Crohn's disease,” and “ulcerative colitis.” Here, we evaluate results obtained using the following equation search (“dairy products” [MeSH Terms] OR (“dairy” [All Fields] AND “products” [All Fields]) OR “dairy products” [All Fields] OR (“dairy” [All Fields] AND “product” [All Fields]) OR “dairy product” [All Fields]) OR (“probiotics” [MeSH Terms] OR “probiotics” [All Fields]) OR (“microbiota” [MeSH Terms] OR “microbiota” [All Fields]) AND ((“inflammatory bowel diseases” [MeSH Terms] OR (“inflammatory” [All Fields] AND “bowel” [All Fields] AND “diseases” [All Fields]) OR “inflammatory bowel diseases” [All Fields] OR (“inflammatory” [All Fields] AND “bowel” [All Fields] AND “disease” [All Fields]) OR “inflammatory bowel disease” [All Fields]) OR  OR “ulcerative colitis” [All Fields] OR (“colitis” [All Fields] AND “ulcerative” [All Fields]) OR “colitis, ulcerative” [All Fields]) OR (“crohn disease” [MeSH Terms] OR (“crohn” [All Fields] AND “disease” [All Fields]) OR “crohn disease” [All Fields]) AND Clinical Trial [ptyp]). One hundred and thirteen original articles matching these criteria were initially selected, although only those articles that included specific LAB and bifidobacteria results (sixty) were later considered for the review and separated into four major topics: general aspects of probiotics in inflammatory bowel diseases, LAB, and bifidobacteria in Crohn's disease, in UC and on other inflammatory bowel diseases. In addition, we focused on the possible probiotic mechanism of action in IBD. ω-3 fatty acids, among other nutrients [Nutrition seems to play a causal role in both UC and CD –17. In tutrients . No specutrients –21. On tutrients .The efficacy of some probiotics to improve IBD patients' quality of life has been recently reported –28. The  E. coli, coliforms, and bacteroides in the colon [There is evidence that commensal enteric bacteria and their products create a local environment that affects the course of IBD . These hhe colon . In thishe colon –33.An individualized diet together with the use of a suitable probiotic may be the best strategy for improving IBD patients' quality of life. The specific knowledge of the mechanisms of action of probiotics would be a helpful tool to design an efficient and specific therapy to improve the specific disease symptoms in IBD.Some of the proposed mechanisms by which probiotics may exert beneficial effects are (1) the production of SCFAs and lactate, which inhibit the growth of potentially pathogenic organisms and have an anti-inflammatory effect on the gut; (2) the increased transit time by the net flow of water from the blood to the intestinal lumen, which influences the adherence of bacteria to the intestinal wall; and (3) the reduced production of noxious substances that may contribute to the pathogenesis of IBD .An altered epithelial barrier function contributes to intestinal inflammation. Moreover the gut microbiota plays a fundamental role in the maturation of the host's innate and adaptive immune responses . The regα), interleukin (IL)-1, and IL-6 [The induction of tolerance or intestinal inflammation depends on a host's ability to distinguish between pathogenic invaders and harmless resident organisms . In IBD,and IL-6 , 37, 38. Lactobacillus rhamnosus GG (LGG) prevents the degradation of Iκβ-α, consequently suppressing nuclear factor kappa B (NF-κB) activation and, in this way, preventing the production of proinflammatory cytokines [ Bifidobacterium and Lactobacillus strains interact with TLR2 and/or TLR9 to enhance the intestinal epithelial barrier function and to facilitate Treg cell conversion via CD103+ DC [On the other hand, apical TLR9 activation in intestinal epithelial cells byytokines , 38. HowD103+ DC , 37. The L. acidophilus X37, L. paracasei Z11, L. casei CRL431, LGG, B. longum Q46, B. bifidum Z9, B. breve 20091, and B. bifidum 20082a decreased IL-12 and TNF-α concentrations in culture supernatants and inhibited the Th1 skewing effect induced by strong stimulatory lactobacilli. This immunoinhibitory effect of bifidobacteria is TLR2-dependent and NOD2-independent [ Bifidobacterium breve CNCM I-4035 also provides immunomodulatory effects on human intestinal DCs, mediated by cytokines [Thus, Zeuthen et al.  reportedytokines , 40.Bacteroides supports T helper (Th) and regulatory T (Treg) cell polarization in a TLR2-dependent manner through the recognition of polysaccharide A by DCs [ Lactobacillus strains GR-1 and RC-14 promotes a desirable anti-inflammatory environment in patients that are consistent with the putative immunosuppressive role of the expanded CD4+CD25high  T cell population in humans [reg cells through an immunoregulatory response involving IL-10 and transforming growth factor beta (TGF-β) [reg) and direct interaction with IgA, probiotics attenuate the immune response against commensal bacteria [A by DCs . The shon humans . Similar (TGF-β) . Via botbacteria . More rebacteria  describebacteria .reg cell-mediated mucosal inflammation and increasing production of IL-10 and TGF-β. This inflammation reduction may prevent colitis. However, further research should be performed with new LAB strains in experimental models of IBD and humans with either CD or UC. Also, the use of combinations of different probiotics should be studied.In summary, a specific probiotic bacterial strain could improve the state of the intestine by facilitating epithelial barrier functions, inhibiting TCD is a chronic inflammatory condition of the gastrointestinal tract driven by abnormal T cell responses to the intestinal microbiota . TherapyMost of the published controlled trials showed that 5-aminosalicylic acid (5-ASA) is significantly more effective than placebo in preventing relapses of the disease. However, negative results have also been reported , 48. ThePrantera et al.  conducte Lactobacillus johnsonii LA1 in this setting with a randomized, double-blind, placebo-controlled study. Patients were randomized to receive two packets per day of lyophilized L. johnsonii LA1 or a placebo for 6 months, and no other treatment was allowed. The primary endpoint was endoscopic recurrence at six months, with a grade >1 in Rutgeerts' classification or an adapted classification for colonic lesions. Ninety-eight patients were enrolled (48 in the L. johnsonii LA1 group). At 6 months, endoscopic recurrence was observed in 64% of the placebo group and in 49% in the L. johnsonii LA1 group. The endoscopic score distribution did not differ significantly between the L. johnsonii LA1 and placebo groups. The L. johnsonii LA1 did not have a sufficient effect, if any, to prevent the endoscopic recurrence of CD [The use of LGG is not restricted only to adult studies. Bousvaros et al.  conductece of CD . L. johnsonii LA1 on early postoperative endoscopic recurrence of CD. The oral administration of L. johnsonii LA1 in patients with CD failed to prevent early endoscopic recurrence at 12 weeks after ileocecal resection [Additionally, van Gossum et al.  evaluateesection . The use L. johnsonii LA1 and cannot predict the inefficacy of other single strains in future trials [Probiotics differ strongly and it is not possible to extrapolate a positive or negative result from one strain to another strain. Therefore, the ineffectiveness of LGG in the study of Prantera et al.  cannot pe trials . ExtrapoPrebiotics have been associated with increased SCFA, mainly acetate, propionate, and butyrate . Short-cA synbiotic is a regimen whereby probiotics are combined with prebiotics. Chermesh et al.  evaluate Bifidobacterium and Lactobacillus. Prebiotics, such as psyllium, are dietary substances that stimulate the growth and metabolism of protective commensal enteric bacteria. Patients were free to adjust their intake of probiotics or prebiotics throughout the trial. The Crohn's disease activity index (CDAI), International Organization for the Study of Inflammatory Bowel Disease (IOIBD) score, and blood sample variables were evaluated and compared before and after the trial. By the end of therapy, each patient had taken a 4.5 ± 2.4 × 1010 colonic forming-unit (CFU) daily probiotic dose, with six patients taking an additional 7.9 ± 3.6 g daily psyllium dose. Seven patients had improved clinical symptoms following combined probiotic and prebiotic therapy. Both CDAI and IOIBD scores were significantly reduced after therapy. There were no adverse events [Ten outpatients with active CD without a history of operation for CD were enrolled in a clinical study to evaluate the effects of synbiotics. Probiotics mainly comprisede events . This st Bifidobacterium longum and Synergy 1. Their clinical status was scored and rectal biopsies were collected at the start, then again at 3- and 6-month intervals. The transcription levels of immune markers and mucosal bacterial 16S rRNA gene copy numbers were quantified using real-time PCR. Significant improvements in clinical outcomes occurred with synbiotic consumption, with reductions in both CDAI and histological scores. The synbiotic had little effect on mucosal IL-18, interferon γ, and IL-1β. However, significant reductions occurred in TNF-α expression in synbiotic patients at 3 months, but not at 6 months [Finally, Steed et al.  evaluate6 months . The synIn conclusion, the investigation presented provides evidence that synbiotics (pre- and probiotics) have the potential to be developed into acceptable therapies for acute and active CD. More studies are needed to determine whether the synbiotic modulates other anti-inflammatory components of the mucosal microbiota , 59, or α antibodies have been used. However, these treatments present side effects that mean that a significant proportion of patients do not tolerate the existing treatments [UC is a nonspecific colorectal erosive inflammatory condition characterized by inflammation of the mucosa, erosion, and ulceration . Patienteatments .Numerous studies, in both IBD patients and gnotobiotic animals, have noted the influence of the intestinal bacteria on the development and/or exacerbation of UC . MoreoveBifidobacteria-fermented milk (BFM) supplementation may reduce exacerbations of UC through the normalization of the intestinal microbiota . Ishikawα exerts a pivotal role in the pathogenesis of active UC; therefore, inhibiting its secretion in inflamed UC mucosa is a major target for treating the disease and preventing relapse [ B. longum reduced the release of TNF-α and IL-8 compared with the inflamed colonic tissue alone. It is well known that the activation of NF-κB can regulate inflammatory cytokines such as TNF-α, IL-8, and IL-6. Immunohistochemical staining of NF-κB p65 in colonic biopsies from active UC showed many cells with positive nuclear staining, whereas fewer NF-κB-positive cells were found in the lamina propria after the tissues were cocultured with either B. longum or dexamethasone, which indicates that B. longum can inhibit NF-κB activation in lamina propria cells [TNF- relapse . Cocultuia cells . L. acidophilus strain La-5 and B. animalis subsp. lactis strain Bb-12, was tested for the maintenance of remission in patients with left-sided UC, in a 1-year, prospective, randomized, double-blind and placebo-controlled trial [Probio-Tec AB-25, a mixture ofed trial . The safed trial .κB, decrease the expression of the proinflammatory cytokines TNF-α and IL-1β, and increase the expression of the anti-inflammatory cytokine IL-10 [The use of BIFICO  in combination with sulphasalazine (SASP) and glucocorticoid exerts some beneficial effects in preventing the relapse of UC . The admne IL-10 . B. infantis 35624 (1 × 1010 CFU) for six weeks to patients with mild- to moderate-active UC, during concurrent treatment with 5-ASA, significantly reduced plasma C-reactive protein (CRP) levels versus the placebo-treated controls [α levels, no significant differences were observed between the group that received the probiotic strain and the placebo group, or in the UC patients before treatment and after treatment. Regarding plasma IL-6, Groeger et al. [In the same way, the administration ofcontrols . Howeverr et al.  found a r et al. . L. rhamnosus, which is represented in the bowel of healthy individuals [10 viable bacteria/day), 87% and 80% for the mesalazine group (2.400 mg/day), and 94% and 84% for the combined treatment  [The most studied probiotic in clinical trials isividuals . In agreividuals  studied alazine) .10 CFU of L. delbrueckii and L. fermentum, together with 2,400 mg/day of sulfasalazine, during 8 weeks, to UC patients with chronic diarrhea, inhibited the extent of inflammation, prevented mucosal injury, and alleviated colitis [ TNF-α. Regarding NF-κB p65 levels, the UC patients showed the more activated NF-κB p65 protein, whereas the lowest level was observed in the probiotic group [The oral administration of Lacteol , a probiotic preparation that contains 1 × 10 colitis . One inf colitis . Lacteolic group . L. reuteri ATCC 55730 (as an enema solution containing 1 × 1010 CFU) for 8 weeks in addition to standard oral mesalazine resulted in a significant decrease in the Mayo DAI score (Mayo Disease Activity Index-DAI) compared with the children that received the corresponding placebo. In addition, all of the children on L. reuteri had a clinical response, whereas only 53% of the children on the placebo responded. Clinical remission was achieved in 31% of the L. reuteri group and in no children of the placebo group. At the posttrial the rectal mucosal expression levels (determined by RT-PCR in biopsy samples) of IL-10 were significantly increased, whereas a significant decrease was found in the levels of IL-1β, TNF-α, and IL-8, only in the L. reuteri group [In children with distal active UC, rectal administration ofri group . L. casei DG on colonic-associated microbiota, mucosal cytokine balance, and TLR expression in patients with mild left-sided UC. The patients were divided into three groups: the first group received oral 5-ASA alone, the second group received oral 5-ASA plus oral L. casei DG (8 × 108 CFU), and the third group received oral 5-ASA and rectal L. casei DG (8 × 108 CFU). A significant improvement of the histological disease severity scores was found in patients receiving the probiotic strain by the oral or rectal route of administration. Nevertheless, oral supplementation with L. casei DG did not have a significant effect on the counts of Enterobacteriaceae or Lactobacillus. However, the occurrence of Lactobacillus and Enterobacteriaceae cultured from biopsy specimens was increased and decreased, respectively, in the group that took the probiotic rectally. Moreover, the rectal administration of L. casei DG significantly reduced TLR-4 and IL-1β levels and significantly increased mucosal IL-10 [Additionally, D'Incà et al.  evaluateal IL-10 .B. longum plus Synergy 1), for a period of one month to patients with active UC, improved the full clinical appearance of chronic inflammation [α and IL-1α were significantly reduced after treatment. In addition, the levels of bifidobacteria, determined by quantitative PCR, increased 42-fold in the synbiotic group but only 4.6-fold in the placebo group [Probiotic therapy can be improved through combination with a prebiotic (a nondigestible oligosaccharide that is absorbed in the upper gut). This combination is known as a synbiotic . In a doammation . In thisbo group .9 CFU of B. longum  once daily; the prebiotic group was prescribed 4.0 g of psyllium to be taken twice daily. The synbiotic group simultaneously underwent probiotic and prebiotic therapies. The doses of aminosalicylates and prednisolone for UC treatment remained the same throughout the trial in all groups [From this study, it is clear that synbiotic positively affects the chronic inflammation associated with UC. The comparison of the effectiveness of probiotics or prebiotics with that of synbiotic therapy was conducted by Fujimori et al. . They del groups . At the l groups . Bifidobacterium breve strain Yakult (BbY) and galactooligosaccharide (GOS), as a synbiotic, were evaluated by Ishikawa et al. [9 CFU/g) and 5.5 g of GOS once/day. The control group comprised patients treated as usual . After one year of intervention, the endoscopic scores of the synbiotic group were significantly lower than in the control group. In addition, the amounts of MPO in the lavage solution significantly decreased in patients with active UC after synbiotic treatment. Fecal bacteria analyses showed significant differences in the number of Bacteroidaceae before and after the synbiotic treatment in UC. Moreover, fecal pH was significantly lower after the synbiotic treatment [The beneficial effects of livea et al. . Patientreatment . Lactobacillus , three strains of Bifidobacterium , one strain of Streptococcus salivarius subsp. thermophilus, and cornstarch. VSL#3 is capable of colonizing the gut and significantly decreases fecal pH in UC patients that are intolerant or allergic to 5-ASA [β-alanine by the colonic bacteria. The use of 2.25 g of balsalazide  plus 3 g of VSL#3 achieved remission faster than balsalazide or mesalazine. Moreover, balsalazide plus VSL#3 showed significant superiority in improving well-being and bowel frequency and endoscopic and histological scores were significantly better in the group of patients who received balsalazide/VSL#3 compared with the patients who received mesalazine at the end of the treatment [The probiotic preparation VSL#3 has been extensively used. VSL#3 contains four strains ofto 5-ASA . Furtherto 5-ASA . Additioto 5-ASA . Balsala12 bacteria per day) for 8 weeks was safe and able to reduce the UCDAI (Ulcerative Colitis Disease Activity Index) scores. Moreover, VSL#3 improved rectal bleeding and seemed to reinduce remission in relapsing UC patients, although these parameters did not reach statistical significance [In addition to this study, Tursi et al.  conducteificance .12 bacteria daily in two divided doses for 6 weeks, resulted in a combined induction of remission/response rate of 94% in patients who completed the study. It is important to highlight that the authors reported no adverse events other than mild bloating [ S. salivarius subsp. thermophilus and B. infantis were detected by PCR/denaturing gradient gel electrophoresis, in association with biopsies collected after (but not before) treatment with VSL#3 in the case of 3 patients in remission [Bibiloni et al.  describebloating . In addiemission .12 bacteria/day) or an identical placebo associated with concomitant steroid induction treatment. Remission was achieved in 92.8% of the children treated with VSL#3 and IBD conventional therapy and in 36.4% of the patients treated with placebo and IBD conventional therapy. Furthermore, 21.4% of the patients receiving VSL#3 treatment and 73.3% receiving the corresponding placebo  relapsed within 1 year of follow-up. Regarding the endoscopic and histological scores, at 6 months and 12 months, they were significant lower in the VSL#3 group. It is important to emphasize that no side effects or significant changes from baseline values in any of the laboratory parameters examined were reported that could be attributed to treatment with either VSL#3 or placebo [In addition, the efficacy of VSL#3 in the induction and maintenance of remission and their safety and tolerability in children has been evaluated in a prospective, 1-year (or until relapse), placebo-controlled, double-blind study conducted by Miele et al. . Patient placebo .α and enhanced the expression of anti-inflammatory cytokine such us IL-10, likely through the inhibition of NF-κB activation.In conclusion, the use of probiotics and/or synbiotics has a positive effect in the treatment of UC and in the maintenance of remission periods. Probiotics and/or synbiotics reduced the expression of proinflammatory cytokines such us TNF- L. acidophilus (La-5) and B. lactis (Bb-12) for 4 weeks increased the number of lactobacilli and bifidobacteria in the UC/IPAA patients and remained significantly increased one week after the intervention. Moreover, involuntary defecation, leakage, abdominal cramps the need for napkins, fecal number, fecal consistency, fecal mucus, and urge to evacuate stools were significantly decreased/improved during the intervention period in the UC/IPAA patients [Pouchitis is a common troublesome condition in surgical patients with ileal-pouch-anal-anastomosis (IPAA)  and is apatients . Enterobacteriaceae within the mucosa during the VSL#3 treatment. This fact indicates that remission maintenance during probiotic therapy is associated with the restoration of parts of the normal pouch biota [The effects of the administration of VSL#3 (6 g/day) on patients with antibiotic therapy-induced pouchitis in remission have been studied by Kühbacher et al. . The autch biota . S. thermophilus; however, no modification of Bacteroides, clostridia, coliforms, and enterococci was found, suggesting that the beneficial effect was not mediated by the suppression of the endogenous microbiota. These data indicate that the efficacy of VSL#3 may be related to increased concentrations of protective bacteria and further support the potential role for probiotics in IBD therapy [Similarly, oral administration of high doses of VSL#3 was effective in the treatment of active mild pouchitis. The authors reported that treatment with VSL#3 significantly improved clinical, endoscopic, and histologic parameters on the PDAI (Pouchitis Disease Activity Index), with complete remission in almost 70% of the patients . The mic therapy .12 bacteria/day) or no treatment (control group) for 12 months. The patients treated with the probiotic showed a slight but significant reduction in PDAI scores after 3 months of treatment compared with baseline. This difference was maintained at 6 and 12 months of follow-up. Moreover, the data obtained by Pronio et al. [ Foxp3 mRNA, a transcription factor needed for the generation and function of regulatory CD4+CD12+T cells and CD4+CD25+T cells that control the immune response to self and foreign antigens and are involved in oral tolerance. Furthermore, tissue samples showed a significant reduction in IL-1β mRNA expression. The authors concluded that the administration of probiotics after IPAA in patients without signs or symptoms of acute pouchitis induces an expansion of the associated regulatory cells [In addition to these studies, Pronio et al.  carried o et al.  showed try cells .IBD and irritable bowel syndrome (IBS) can be considered as different pathologies. IBD is recognized as an organic bowel disorder while IBS is a functional bowel disorder, although some particular cases in both disorders may display similar symptoms. Therefore, distinguishing clinical manifestations may be sometimes difficult , 81. IBS Lactobacillus sp. HY7801, Lactobacillus brevis HY7401, and Bifidobacterium longum HY8004) on seventy-four IBS patients through clinical parameters and 1H nuclear magnetic resonance- (NMR-) based metabolomics from peripheral blood. This study reported decreased glucose and tyrosine levels and increased lactate in sera of patients but not in healthy volunteers. They argued that this increase in lactate in blood might be caused by intestinal microbiota that produce lactate through fermentation because of increased populations of intestinal LAB after probiotic administration. They further related the low serum glucose levels to elevated glycolysis in the body's attempt to accommodate the higher energy demand caused by small nutrient absorption [Hong et al.  evaluatesorption . They alsorption . Bifidobacterium longum W11, one of the most representative species of gut microbiota, and oligosaccharides, which exert a positive effect on intestinal motility and favor the development of bifidobacteria in the gut lumen [Furthermore, Dughera et al.  confirmeut lumen . Althougut lumen , 85.Cholangitis is an infection of the common bile duct, the tube that carries bile from the liver to the gallbladder and intestines. It is usually caused by a bacterial infection, which can occur when the duct is blocked, such as a gallstone or tumor. The infection causing this condition may also spread to the liver . L. acidophilus, L. casei, L. salivarius, L. lactis, B. bifidum, and B. lactis) have been evaluated on the liver biochemistry or function and symptoms in primary sclerosing cholangitis (PSC) patients with IBD that were receiving ursodeoxycholic acid (UDCA) maintenance therapy [L. casei Shirota, 3 g/day) provided benefits for both IBD and PSC. They suggested that bacterial microbiota and gut inflammation are closely associated with the pathogenesis of IBD-related PSC. This suppression of bowel inflammation and maintenance of bacterial homeostasis may be important for treating PSC [The effects of a probiotic mixture , metabolomics, transcriptomics, and proteomics analyses of biopsies.Beyond understanding the molecular mechanisms, further studies to evaluate the best dose-response-effect of probiotics are recommended, including following up with patients after the probiotic intervention to evaluate the persistence of beneficial effects.Finally, determining the effect of fermented dairy products on the development and maintenance of the disease will also require specific clinical trials.


Epithelial ovarian cancer (EOC) is the most deadly of the gynecological cancers. New approaches and better tools for monitoring treatment efficacy and disease progression of EOC are required. In this study, metabolomics using rapid resolution liquid chromatography mass spectrometry was applied to a systematic investigation of metabolic changes in response to advanced EOC, surgery and recurrence. The results revealed considerable metabolic differences between groups. Moreover, 37, 30, and 26 metabolites were identified as potential biomarkers for primary, surgical and recurrent EOC, respectively. Primary EOC was characterized by abnormal lipid metabolism and energy disorders. Oxidative stress and surgical efficacy were clear in the post-operative EOC patients. Recurrent EOC patients showed increased amino acid and lipid metabolism compared with primary EOC patients. After cytoreductive surgery, eight metabolites  corrected towards levels of the control group, and four  went back again to primary EOC levels after disease relapse. In conclusion, this study delineated metabolic changes in response to advanced EOC, surgery and recurrence, and identified biomarkers that could facilitate both understanding and monitoring of EOC development and progression. Epithelial ovarian cancer (EOC) continues to be one of the most frequently diagnosed malignancies worldwide, and is the most deadly of the gynecological cancersCancer cells possess a fundamentally altered metabolism that enables tumorigenicity and malignancy7891011121310111213In this study, four batches of plasma samples were collected from the following patients: 35 pairs of EOC patients at the time of diagnosis (primary EOC patients) and after cytoreductive surgery (post-operative EOC patients), 35 matched controls, and 35 matched recurrent EOC patients. Metabolic profiling of these samples was performed by rapid resolution liquid chromatography mass spectrometry (RRLC/MS). First of all, potential biomarkers were identified for the primary, surgical and recurrent EOC, respectively. Then, metabolic changes of primary EOC biomarkers were monitored in the post-operative and recurrent EOC patients. Our ultimate goals were (1) to investigate metabolic alterations that underlie the responses to advanced EOC, surgical intervention and disease relapse; and (2) to discover potential biomarkers that could monitor EOC surgical efficacy and disease relapse.P > 0.05). Detailed clinical characteristics for the participants are listed in Thirty-five plasma samples from primary EOC patients, 35 from the same EOC patients after surgery, 35 from relapsed EOC patients, and 35 from controls were included in this study. The controls were age- and menopause-matched with primary EOC patients, and the recurrent EOC patients were age-, menopause- and stage-matched with primary EOC patients. According to the International Federation of Gynecology and Obstetrics (FIGO) staging system, 31 of the EOC patients were classed as stage III and four as stage IV. All EOC patients enrolled in this study had received cytoreductive surgery followed by platinum-based chemotherapy. There were no statistical differences observed for age, menopause, stage, weight, lymph node status, histology type, and histology differentiation among the four groups , fatty acids, amino acids, and bile acids. The metabolites, including biochemical characteristics, statistical information and metabolic pathways, are detailed in Typical chromatograms of our plasma samples are shown in P < 0.05. Post-operative EOC patients were characterized by increased fatty acids, amino acids, and uric acid, and decreased LPCs, l-kynurenine, and indoxylsulfuric acid  and amino acids, and decreased 2-octenoic acid and creatine  and amino acid-related metabolites  were remarkably increased in relapsed EOC patients compared with the primary EOC patients. Further significant alterations in recurrent EOC patients were observed within lipid metabolism, as indicated by significantly increased levels of LPCs, LPEs and fatty acids. Altered pathways of amino acids and lipids have been suggested to contribute to platinum resistance in ovarian cancer cellsl-tryptophan to l-kynurenine by indoleamine-pyrrole 2,3-dioxygenase (IDO) has been well characterized in ovarian cancer33l-kynurenine did not recover towards primary EOC levels in recurrent EOC patients. Therefore, our findings suggest that while IDO inhibitors may be promising for first-line therapy, they may not be applicable to resistant ovarian cancer.Recurrent EOC patients are usually characterized by one of the biggest mysteries in cancer research, chemotherapy resistance, which accounts for most therapeutic failures and eventual deathImportantly, the levels of 2-octenoylcarnitine, 2-octenoic acid, 19,20-DiHDPA and hydroxyphenyllactic acid in the post-operative EOC patients trended towards recovering to levels of the control group but then trended towards primary EOC levels after disease relapse. As discussed above, recovery of these metabolites in the post-operative EOC patients could be attributed to surgical efficacy. Their recurrence in relapsed EOC patients is most likely caused by resurgent tumor proliferation. These four biomarkers are excellent potential biomarkers for monitoring EOC progression.To the best of our knowledge, this study is the first to examine plasma metabolic changes in response to EOC surgery and recurrence. However, there were several limitations. One is the relatively small sample size in each group, which might prevent the differences in some metabolites from being fully apparent. In addition, a targeted and quantitative analysis of the potential biomarkers is needed to advance clinical translation. For future research, we are collecting a larger sample cohort and performing a targeted metabolomic investigation into these biomarkers.l-tryptophan metabolism. The phenomena of oxidative stress and surgical efficacy are clear in the metabolic profiles of post-operative EOC patients. Metabolic signatures in recurrent EOC patients show increased amino acid and lipid metabolism compared with primary EOC patients. In all, this study highlights a number of potential EOC biomarkers that could facilitate both understanding and monitoring of advanced EOC, its remission and progression.In summary, we present a holistic view of the plasma metabolic changes related to advanced EOC at diagnosis, surgery and relapse. Specific metabolite markers have been identified that could be used to monitor surgery efficacy and disease relapse. Primary EOC is characterized by abnormal lipid metabolism, energy disorders and altered g for 10 min, and the supernatant was collected and frozen at −80 °C until analysis.The present experiments were conducted in accordance with the 1975 Declaration of Helsinki. The protocol in this study was approved by the Ethics Committee of the Tumor Hospital at Harbin Medical University, Harbin, China. Plasma samples were collected by the Department of Gynecology of the Tumor Hospital at Harbin Medical University between January 2012 and July 2013. All the participants gave informed written consent. The inclusion criteria and exclusion criteria were as follows: (1) participants suffering from metabolic, liver, or kidney diseases or any other cancers were excluded; (2) controls were defined as uterine fibroid patients or benign ovarian tumor patients; (3) primary EOC patients and controls were not taking any medications, and their diagnosis was confirmed by histopathology after surgery; (4) blood samples from post-operative EOC patients were collected within 3–7 days of cytoreductive surgery; (5) recurrent EOC patients were confirmed by follow-up blood tests, physical examinations, and imaging tests. After fasting for 12 h, 5 mL of whole blood was collected from each patient into an EDTA tube. Fresh blood was centrifuged at 1,000 ×To ensure the stability and repeatability of the RRLC/MS systems, QC samples were used in both electrospray ionization positive (ESI+) and negative (ESI−) modes. Pooled QC samples were prepared from a mixture of all plasma samples. One QC sample was run after every twelve samples.g for 10 min at 4 °C. The supernatant was transferred into a clean vial, and evaporated until dry, under reduced pressure. The residue was dissolved in 100 μLof acetonitrile/water , vortex-mixed for 1 min, and then centrifuged at 12,000 ×g for 15 min at 4 °C. The supernatant was then placed into the sample vial for analysis.In order to ensure a balance in sample size across groups over time, sample pretreatment and RRLC/MS analysis were performed using the blocked randomization methodA 10 μL aliquot of the pre-treated sample was injected into a 3.0 × 100 mm (1.8 mm) ZORBAX SB-C18 column  for RRLC . The mobile phase for ESI+ was a mixture of acetonitrile containing 0.1% formic acid (A) and water containing 0.1% formic acid (B). The mobile phase for ESI− was a mixture of acetonitrile (A) and water (B). A linear mobile phase gradient was used as follows: 2% A, held for 1 min; 1–18 min, increased to 98% A; 18–21 min, held at 98% A; 21–21.1 min, decreased to 2%A; and 21.1–28 min, held at 2% A. The mobile phase flow rate was 0.3 mL/min at 40 °C.m/z.Mass spectrometry was performed with an Agilent 6530-QTOF (Agilent Technologies) operating in both ESI+ and ESI− modes. The capillary voltage was set as 4.0 kV for ESI+ mode and 3.5 kV for ESI− mode. Nitrogen was used as the desolvation gas at a flow rate of 10 L/min. The desolvation temperature was 350 °C. Centroid data were collected in full scan mode from 50 to 1000 m/z) values, and peak intensities. CAMERA in R was then used to annotate isotope peaks, adducts and fragments in the peak listsThe XCMS package was used to preprocess the raw data in the R platform, including peak detection, peak matching, matched filtration, and retention time alignmentt-test was used to further validate the significance of each metabolite. Potential metabolic biomarkers were selected using VIP > 1 and P < 0.05. To visualize metabolite changes across advanced EOC, surgery and recurrence, the standardized metabolite level in the four groups was plotted in an error bar chart.The preprocessed data was exported to SIMCA-P 11.5 software for multivariate analysis. Principal component analysis (PCA) was first used to detect separation trends and outliersHow to cite this article: Ke, C. et al. Metabolic phenotyping for monitoring ovarian cancer patients. Sci. Rep. 6, 23334; doi: 10.1038/srep23334 (2016).


Plasmodium falciparum in India has historically travelled from northeast India along the Myanmar border. The treatment policy for P. falciparum in the region was, therefore, changed from chloroquine to artesunate (AS) plus sulphadoxine-pyrimethamine (SP) in selected areas in 2005 and in 2008 it became the first-line treatment. Recognizing that resistance to the partner drug can limit the useful life of this combination therapy, routine in vivo and molecular monitoring of anti-malarial drug efficacy through sentinel sites was initiated in 2009.Anti-malarial drug resistance in Between May and October 2012, 190 subjects with acute uncomplicated falciparum malaria were enrolled in therapeutic efficacy studies in the states of Arunachal Pradesh, Tripura, and Mizoram. Clinical and parasitological assessments were conducted over 42 days of follow-up. Multivariate analysis was used to determine risk factors associated with treatment failure. Genotyping was done to distinguish re-infection from recrudescence as well as to determine the prevalence of molecular markers of antifolate resistance among isolates.P. falciparum cleared parasitaemia within 24 hours of treatment, but eight, including three patients who failed treatment, remained parasitaemic on day 3. Risk factors associated with treatment failure included age < five years, fever at the time of enrolment and AS under dosing. No adverse events were reported. Presence of dhfr plus dhps quintuple mutation was observed predominantly in treatment failure samples.A total of 169 patients completed 42 days of follow-up at three sites. The crude and PCR-corrected Kaplan-Meier survival estimates of AS + SP were 60.8% (95% CI: 48.0-71.4) and 76.6% (95% CI: 64.1-85.2) in Gomati, Tripura; 74.6% (95% CI: 62.0-83.6) and 81.7% (95% CI: 69.4-89.5) in Lunglei, Mizoram; and, 59.5% (95% CI: 42.0-73.2) and 82.3% (95% CI: 64.6-91.6) in Changlang, Arunachal Pradesh. Most patients with AS + SP treatment failure was widespread in northeast India and exceeded the threshold for changing drug policy. Based on these results, in January 2013 the expert committee of the National Vector Borne Disease Control Programme formulated the first subnational drug policy for India and selected artemether plus lumefantrine as the new first-line treatment in the northeast. Continued monitoring of anti-malarial drug efficacy is essential for effective malaria control. Plasmodium falciparum was a major contributor to global resurgence of malaria in the 20th Century. Southeast Asia (SEA) has been the focus of drug resistance for all anti-malarials with loss of valuable drugs from time to time. Chloroquine resistance in P. falciparum malaria was first reported in 1957 in SEA region, later followed by sulphadoxine-pyrimethamine resistance after ten years [P. falciparum[Plasmodium vivax, another parasite that contributes equally to human malaria in the country.Anti-malarial drug resistance, particularly in en years . India sen years . HoweverP. falciparum malaria in the country is based on artemisinin combination therapy (ACT) as per the recommendation of World Health Organization (WHO) [dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes, both coding for essential enzymes in the folate biosynthesis pathway, led to resistance to antifolate drugs [Since 2005, treatment of uncomplicated on (WHO) . The ACTon (WHO) . Howeveron (WHO) , which tte drugs .Recent reports on artemisinin resistance in the SEA region  also reqdhfr plus dhps) are potential early warning signs; the same were also monitored for the study sites which prompted more focus in these study areas.The Indian northeastern region has varied ecological diversity, inaccessible areas where vector control measures as well early treatment and diagnosis facilities are difficult to assess. Malaria control remains a challenge in these areas. ACT was introduced in the study areas in 2007 and since 2009 continuous monitoring of recommended anti-malarials has been done through nationwide sentinel site system. Also, partner drug resistance markers ; from August to September 2012 in Tripura ); from September to October 2012 in Arunachal Pradesh . The study sites  cases.The state contributes 1.3% to the country’s malaria burden . The peaPlasmodium falciparum is a major malaria parasite in this region, causing 70 to 90% of malaria infections. The hot and humid climate in the region is ideal for survival and multiplication of malaria vectors [Tripura is one of the states in the northeastern region which shares a long international border with Bangladesh. The hilly and undulating terrain and the movement of people across the border have led to persistence of malaria in villages near the border. The regions adjacent to the Indo-Bangladesh border are mostly covered with thick forests and have poor communication and health infrastructure.  vectors .P. falciparum has increased from 56 to 64% in the last five years.Changlang district in the state of Arunachal Pradesh is bounded by Assam and Arunachal Pradesh in the north and Myanmar in the southeast. The state contributes 1.1% of the country’s malaria burden . The capin vivo monitoring was followed [This was an open-label, one-arm prospective study of clinical and parasitological responses after administration of ACT treatment and WHO protocol for followed .Informed, written consent was obtained from enrolled adults and from a legal guardian of each child enrolled. The study protocol was approved by the Institutional Ethics Committee of the National Institute of Malaria Research (NIMR) in New Delhi. These studies were part of monitoring and surveillance studies jointly carried out by NIMR and National Vector Borne Disease Control Programme (NVBDCP) with added quality assurance, monitoring and molecular markers.P. falciparum with parasitaemia between 500 and 100,000 asexual parasites/μl blood, absence of other febrile conditions and informed consent. Patients having severe malnutrition as per WHO guidelines, severe malaria or danger signs and inability to come for follow-up visits were excluded from the study.Adults and children over 6 months presenting with fever (axillary temperature ≥ 37.5°C) at visit or a history of fever for the preceding 24 hours were included in the study. Other criteria for inclusion were mono-infection with P. falciparum received artesunate (AS) + SP (AS 4 mg/kg for three days plus SP 25/1.25 mg/kg single dose on day 0) and primaquine (PQ) (0.75 mg/kg) on last day of treatment. The treatment was directly observed on all three days and used quality assured drugs through state government supply which consisted of manufacturers (site name in brackets): Medicamen Biotech Ltd, India (Lunglei), Medico Remedies Pvt Ltd, India (Gomati), ZEST Pharma, India (Changlang). These drugs are supplied in the trade name of ‘Antimalarial combi blister pack’. All drugs were used within their expiry period and batch number and expiry dates were recorded in each case record form (CRF). The dosing was based on age with five categories of combi blister packs with age category < one year, one to four years, five to eight years, nine to 14 years and >15 years (adult pack). The study followed the standard WHO protocol for assessment of therapeutic efficacy of anti-malarial drugs for uncomplicated falciparum malaria for moderate transmission [msp1, msp2 (merozoite surface protein) and glurp (glutamate rich protein) gene loci of pre- and post-treatment sample pairs. The outcome of treatment with PCR correction was based on the number of true recrudescence excluding cases of novel infections. In addition, pfdhfr and pfdhps mutations for partner drug resistance were used as molecular markers to analyse different samples.Patients with uncomplicated smission . On enroThick and thin blood films were collected and stained with Giemsa. Slides were examined on day 0 by experienced microscopist(s) for species identification and quantification of parasites. Slides were also prepared on days 1, 2, 3, 7, 14, 21, 28, 35, and 42 to determine asexual and sexual parasite density by counting the number of parasites against 200 white blood cells (WBCs), and were expressed assuming WBC count to be 8,000/microlitre. A slide was considered negative when counting 1,000 WBC in thick smear did not show asexual parasites. All the slides were cross-checked at NIMR, Delhi.msp2 or glurp. The third marker analysed was msp1 to differentiate recrudescence from new infections [pfdhfr gene and pfdhps gene at codon 436, 437, 540, 581, 613 were also analysed in the day 0 samples.Paired blood samples of patients collected on day 0 and the day of recurrent parasitaemia (14–42 days) were analysed sequentially starting with the highest discriminatory marker, fections . A new ifections . In addimsp1 , two allelic families of msp2 (Fc 27 and Ic) and glurp. Pfdhfr and pfdhps gene products were PCR amplified using earlier reported methods [dhfr and dhps gene. Applied Biosystem thermocycler was used for all PCR amplification reactions. Digested PCR product (5–8 microlitre) was analysed on 1.5% agarose gel containing ethidium bromide (0.5 μg/ml) and 0.5X TBE running buffer (pH 8.0). PCR products were visualized under UV transilluminator (280 nm) and digitally captured with the help of gel documentation system (Alpha Imager EP). Molecular sizes of PCR fragments were calculated using gene tool .Genomic DNA was isolated from blood spots using QIAamp DNA minikit, Germany. Genotyping PCR assays were carried out following the protocols reported earlier . Separat methods  and thenThe case record forms were completed for each patient and all the clinical and parasitological data from days 0 to 42 were recorded. The data were entered in WHO software  and bothAll the patients with ETF or LTF were treated with oral quinine in standard dose of 10 mg/kg body weight, three times for seven days. CRFs were used to capture adverse events, if any.P. falciparum were categorized as being ≥ 87.5%, < 87.5 to 75% and < 75% of the recommended dose per kg. The parasite clearance time (PCT) was recorded in CRF till day 2 of treatment, although slides were made on day 3 as well. However the delayed PCT (PCT ≥ 72 hours) was calculated on day 3 based on the mean PCT up to day 2 ± SD. Kaplan-Meier survival analyses of treatment failure and parasite clearance were conducted with and without the results of the PCR-based identification of the parasitaemia that resulted from post-treatment re-infection. Log-risk models were used to evaluate the multivariate associations observed between risk factors and endpoints. Multivariate analysis was based on full models that included age, sex, fever at enrolment, level of parasitaemia at enrolment, infection with a parasite that harboured any of the investigated mutations in dhfr or dhps, and AS dose, as well as the interactions between age and AS dose, age and level of parasitaemia on enrolment, and between presence of mutations and level of parasitaemia on enrolment. A complete case (per protocol) analysis was performed. A strategy of backward elimination was followed in which p-value of < 0.10 and < 0.15, respectively, were used as the cut-offs in eliminating individual factors and interaction terms as statistically significant risk factors for treatment failures. All data analysis was performed using version 7.2 of a software package developed by WHO’s Global Malaria Programme for evaluating therapeutic efficacy or SPSS version 14.Patient and demographic variables that could be associated with treatment failure were investigated. Using the body weights recorded on day 0, the doses of AS given to patients with P. falciparum receiving AS + SP treatment on first three days and PQ on last day of treatment were enrolled from Lunglei district in Mizoram (71), Gomati district in Tripura (77) and Changlang district in Arunachal Pradesh (42), the three far- flung regions of northeast India. Fifteen patients were withdrawn after cross-checking due to mix infection, presence of other species, or out of range parasitaemia at the time of enrolment. Thus, 175 patients were found to be eligible, 169 (96.6%) of whom completed the 42 days of follow-up  of the patients were confirmed as treatment failure, 24 were found to have P. falciparum re-infection and one had P. vivax infection on day 21. These 32 patients who were confirmed to have failed AS + SP treatment came from Lunglei (11), Gomati (15) and Changlang (six)  in < 24 hours, 35.5% (60 of 169) in intervals of 24 to <48 hours, 8.8% (15 of 169) in 48 to < 72 hours and 4.7% (eight of 169) of the patients in ≥ 72 hours, respectively  relative to adults 15 years or older and low administered dose of AS  relative to 3.5 mg/kg or more were associated with higher risk. The risk of failure increased as the dose of AS decreased. However, optimal or higher dose of SP was observed in 90.6% patients. Fever at enrolment was positively and significantly associated with treatment failures. Also, relative risk was highest with the presence of triple or quadruples mutation  and triple mutations (n = 69) were common. The most frequent haplotype was double mutant 108/59 followed by triple mutant 108/59/51. Only five isolates were single mutant while three were found to have quadruple mutation (5.2%).Out of the 190 isolates, 155 could be successfully genotyped for dhps, triple (n = 75) and quadruple mutations (n = 35) were common. Mutations in codon 437 and 540 were most frequent and the most frequent haplotype was triple mutant 540/437/436 followed by quadruple mutant 581/540/437/436  recommended for This study provides the first report on the emerging treatment failure of AS + SP in remote areas of the country, including areas directly across the border from Bangladesh and Myanmar where artemisinin resistance has been confirmed recently. Treatment failure to this ACT is now evident at all the three study sites of northeast India. The failure of AS + SP was likely as the molecular marker of partner drug resistance showed increasing trend since 2009 in the nationwide sentinel site monitoring where 20% random samples were assayed to monitor drug resistance at molecular level. The trend showed increase from single to double mutation in majority of the samples. These studies were part of nationwide sentinel site monitoring system, where 15 sentinel sites are being monitored across the country to ascertain the efficacy of recommended anti-malarials against the predominant species of Plasmodium.Based on the results of this study, the expert committee of the NVBDCP created the first subnational drug policy for India and selected artemether plus lumefantrine as the new first-line treatment in the northeast .ACT treatment failure > 10% is the cost-effective threshold and requires change of treatment policy for malaria along with continuous monitoring in adjacent areas . In the dhps remained wild in majority of the samples, however, increasing trend in dhps point mutations were observed later, particularly in northeastern region. These findings prompted more focus in northeastern region. Thus, three sites at different ecological conditions were selected and efficacy of AS + SP in P. falciparum was studied.Until recently, the efficacy and safety of recommended ACT (AS + SP) was high at multiple sites in the country . The natdhfr and dhps gene was observed from all the three study sites with higher frequency in dhps gene. Sulphadoxine resistance in P. falciparum is associated with mutations at five pfdhps codons; 436ala/phe, 437gly, 540glu, 581gly, and 613ser [pfdhps mutation at codon A436 causes alteration in binding of sulphadoxine followed by sequential mutations at G437, E540, G581, and T/S613 which may cause increase in sulphadoxine resistance [Data on molecular studies for SP resistance are conclusive with increased frequency of quintuple mutation over time. Increased frequency of quadruple mutation in d 613ser . The pfdsistance . Mutatiosistance .pfdhfr codons (108asn + 51ile + 59arg) and two pfdhps codons (437gly + 540glu) [A strong indicator for SP treatment failure is the quintuple mutations in three  540glu) . Quintup 540glu) .in vitro and in vivo has been proposed [Following these findings, further new studies on tracking resistance to artemisinin are being undertaken at two sites in this region. Recently association of K13-propeller polymorphism with artemisinin resistance proposed . FurtherVarious factors including host immunity and pharmacokinetics of the drug play an important role in achieving complete cure or treatment failure in patients. In the present study, younger age category (<five years) relative to adults 15 years or older, fever at enrolment and low administered dose of AS  relative to 3.5 mg/kg or more were associated with higher risk of treatment failure. Negative correlation was observed between the risk of failure with the dose of AS (in mg/kg body weight). Although, the recommended daily dose of AS is 4 mg/kg, 15.6% of treatment failure patients received 3.0 to < 3.5 mg of AS per kg, and 9.4% received < 3.0 mg/kg. Due to variation in body weight within age categories, dosing of drugs based on age category presents the risk of under-dosing or over-dosing. Thus, the criteria to use age rather than body weight for calculating the dose of anti-malarial needed for a patient is the probable cause of suboptimal dosing. However, optimal or higher dose of SP was observed in majority of the patients with only 9.4% patients receiving under-dose of SP. Fever at enrolment was positively and significantly associated with treatment failures. Earlier results of nation-wide sentinel system also observed younger age, fever and low AS dose as the potential risk factors for treatment failure . Partnerin vitro monitoring in the northeast for early warning of this ACT is required.Recently increasing failure rates of artesunate mefloquine in Thailand and dihydroartemisinin piperaquine in Cambodia have been reported ,33. WithThe efficacy of AS + SP was declining in northeast India and exceeded the threshold for changing drug policy. Based on the results of the study, the expert committee of the National Vector Borne Disease Control Programme formulated the first subnational drug policy for India and selected artemether plus lumefantrine as the new first-line treatment in the northeast. Continued monitoring of anti-malarial drug efficacy in northeastern region is essential for effective malaria control.The nation-wide sentinel site monitoring system created in 2009 accelerated the assessment of emerging ACT treatment failure in areas with international borders and is vigilant to detect delayed parasite clearance, if any. Few of the earlier reported limitations of nationwide sentinel system were overcome during 2012. Firstly, follow-up was extended up to 42 days, delayed parasitaemia was monitored by looking at parasite clearance interval of ≥ 72 hours; partner drug resistance markers were analysed in all the isolates. The other limitations include 24 hours sampling for parasite monitoring, as the patients were from distant places. The sample size was relatively low in Changlang district, Arunachal Pradesh. To ascertain the role of partner drug resistance, drug levels of SP could not be done as the samples for drug concentration could not be collected.The authors declare that they have no competing interests.NM, NV and GSS were responsible for the design of the project proposal and monitored progress. NM, KK, BS, JPN and RSB were involved in quality check of data and molecular studies. NM, VD and SP monitored the studies at the site. NM, RR and NKS compiled and performed statistical analysis. NM and NKS wrote the first draft and ACD, GSS, ARA and NV corrected the draft and all authors read and approved the final manuscript.


Aim. Recent evidence suggests that chronic subclinical inflammation plays a key role in the pathogenesis and progression of diabetic nephropathy. Aim of the present study was to investigate possible correlation between the presence and degree of microalbuminuria and markers of inflammation in patients with type 2 diabetes mellitus (DM). Patients-Methods. Eighty patients were enrolled and clinical and laboratory data were recorded. Albumin-creatinine ratio (ACR) was calculated in first-morning urine samples. Serum and urinary tumor necrosis factor-α (TNF-α) levels were determined by ELISA. Results. Forty-five patients had normoalbuminuria, 33 microalbuminuria, and 2 macroalbuminuria. Patients with microalbuminuria were older, with higher glycosylated hemoglobin levels (HbA1c) and they more frequently had diabetic retinopathy, neuropathy, and cardiovascular disease and were on treatment with angiotensin converting enzyme inhibitors (ACEi) and/or angiotensin receptor blockers (ARBs). ACR was significantly correlated with the presence of cardiovascular disease, hypertension, and HbA1c levels and the administration of clopidogrel and ACEi or ARBs. ACR was not correlated with C-reactive protein, fibrinogen, or serum TNF-α levels but had a strong correlation with urinary TNF-α levels. Conclusions. In patients with type 2 DM, urinary, but not serum, TNF-α levels are associated with the presence and severity of microalbuminuria. Hematology and biochemistry were determined by routine techniques using an automated analyser. LDL cholesterol was calculated using the Friedewald formula. Serum C-reactive protein (CRP) levels were evaluated by high sensitivity nephelometry, and exact values were obtained in all patients .α assay. The same specimen was used for urinary cultures and for the measurement of albumin-creatinine ratio, ACR (μg/mg). ACR < 30 μg/mg was defined as normoalbuminuria, 30–300 μg/mg as microalbuminuria, and ACR > 300 μg/mg as macroalbuminuria.First-morning urine samples were collected under sterile conditions. 10 ml of urine were centrifuged at 1500 rpm for 10 min and the supernatant was stored at −80°C until TNF-α assay.Serum samples were separated from clotted blood by immediate centrifugation (1500 rpm for 10 min), aliquoted, and stored at −80°C until TNF-α levels were measured by an enzyme-linked immunoabsorbent assay (ELISA) using commercially available standard kits  in the Laboratory of Kidney Disease Research of the Department of Nephrology at “Hippokratio” General Hospital. The lower detection limit was 0.5 pg/mL. Urinary TNF-α levels were standardized by the amount of creatinine in the urine and expressed as pg/mg urinary creatinine.Serum and urinary TNF- t-test or the Mann-Whitney U test as appropriate. Differences in proportions were tested with the use of the chi-square statistic. Correlations were tested by regression analysis. Not-normally distributed variables were log-transformed before entering regression analysis. Multiple regression analyses with a forward elimination procedure were used to assess the combined influence of variables on ACR values. The calculations were performed using SPSS 17.0, statistical software. A two-tailed P value of 0.05 was considered to be statistically significant.Data are expressed as mean ± SD, median with range or number of patients (%) as appropriate. The significance of differences in means between groups was assessed by Student'sThe demographic, anthropometric, and clinical characteristics of the patients as well as the prescribed drugs are shown in 2. Sixty-one patients (76.3%) were receiving statins, 46 (57.5%) angiotensin converting enzyme inhibitors (ACEi) and/or angiotensin receptor blockers (ARBs), 21 (26.3%) acetylsalicylic acid, and 25 (31.3%) clopidogrel. Diabetes was controlled with oral hypoglycaemic agents alone in 55 (68.8%) patients, insulin in 4 (5%) patients, and a combination of oral hypoglycaemic agents and insulin in 21 (26.3%) patients.Median age was 68 years and 60% of the patients were female. Median duration of diabetes mellitus was 14 years and mean eGFR 76.7 mL/min/1.73 mμg/mg, CRP was 0.52 (0.13–1.9) mg/dL, fibrinogen was 3.28 ± 0.5 g/L, serum TNF-α was 4.28 ± 5.01 pg/mL, and urinary TNF-α was 7.12 ± 8.09 pg/mg creatinine.Laboratory results are shown in P = 0.026), administration of clopidogrel (P = 0.012) and ACEi/ARBs (P = 0.003), and levels SBP . In addition, ACR had a weak, not significant correlation with duration of diabetes , BMI , and the presence of hypertension (P = 0.06) and a borderline correlation with age . No significant association was observed between ACR and the presence of diabetic retinopathy, neuropathy, MBP, and DBP and the administration of statins or acetylsalicylic acid.ACR was significantly correlated with a history of cardiovascular disease (P = 0.01) and had longer duration of diabetes (P = 0.02) and higher glycosylated hemoglobin levels (P = 0.001). In addition, a greater percentage of patients with microalbuminuria had retinopathy (P = 0.02), neuropathy (P = 0.04), and a history of cardiovascular disease (P = 0.02) and were receiving ACEi/ARBs (P = 0.001). ACR showed a positive and significant correlation with glycosylated hemoglobin levels  and a weak negative correlation with eGFR . No association was observed between ACR and LDL and HDL-cholesterol and triglyceride levels.When the presence of microalbuminuria was considered as a categorical parameter, we found that patients with micro- compared to those with normo-albuminuria were older  . Factors included in the multivariate analysis were age, duration of diabetes, BMI, SBP, history of cardiovascular disease, presence of retinopathy, hypertension, glycosylated haemoglobin levels, treatment with clopidogrel, ACEi/ARBs, and eGFR and urinary TNF-α excretion. The variables that were entered into the model had a P value < 0.1 in univariate analysis. Of note, no association was observed between serum and urinary TNF-α levels.ACR had no significant correlation with CRP, fibrinogen, or serum TNF- = 0.01) . MultivaForty-five patients had normoalbuminuria (56%), 33 had microalbuminuria (41%), and only 2 patients had macroalbuminuria. The demographic, anthropometric, and clinical characteristics as well as the administered drugs and the laboratory findings of the patients with normoalbuminuria and microalbuminuria are shown in α (P = 0.001), , , but simThe aim of the present study was to investigate the impact of inflammation during the early phase of diabetic nephropathy. Recent evidence suggests an important role of inflammation in the pathogenesis and progression of diabetic nephropathy . The preAge and duration of diabetes showed a weak positive correlation with the degree of microalbuminuria. Moreover, both parameters were significantly higher in patients with microalbuminuria compared to those with normoalbuminuria. The above results are rather predictable since age and diabetes duration for more than 10 years are well known risk factors for the development of diabetic nephropathy . FurtherThe frequency of diabetic retinopathy was significantly increased in the microalbuminuric compared to the normoalbuminuric patients. This finding is consistent with the definition and the natural history of diabetic nephropathy, in which retinopathy usually precedes or appears simultaneously with the development of albuminuria , 2.In the present study, the degree of microalbuminuria was significantly correlated with systolic blood pressure. It is well known that hypertension and diabetes coexist in the majority of patients, and that systolic blood pressure is a major hemodynamic mechanism of proteinuria in diabetic nephropathy . AlbuminA statistically significant positive correlation was observed between microalbuminuria and glycosylated hemoglobin levels. Moreover, glycosylated hemoglobin levels were higher in microalbuminuric compared to normoalbuminuric patients. The above findings reflect the association between poor glycemic control and the development of diabetic nephropathy , 2. Our Microalbuminuria was associated with the administration of ACEi/ARBs, and, compared to normoalbuminuric, a greater percentage of microalbuminuric patients were on the above drugs. These findings probably reflect the routine prescription of ACEi/ARBs in all diabetic patients with albuminuria due to their well-documented antiproteinuric effect .As mentioned above, recent studies suggest a significant role of inflammation in the pathogenesis and progression of diabetic nephropathy. However, inflammation in diabetic nephropathy is activated by metabolic, biochemical, and haemodynamic disorders which progressively and persistently lead to kidney injury .Inflammation may promote kidney damage through a variety of mechanisms including monocyte migration, complement activation, platelet function regulation, and clearance of cellular debris from inflamed areas . In our Similarly, recent studies have reported higher fibrinogen levels in macro- compared to microalbuminuric patients and controls , while Cα is a polypeptide of 157 amino acid residues and a molecular weight of 17 kDa [α is a nonglycosylated polypeptide and this may be the main difference with mouse TNF-α and with human TNF-β. Both TNF-α and TNF-β act through their receptors, TNF-R1 and TNF-R2, and glycosylated forms of both receptors have been found in the urine [α is produced by monocytes-macrophages and by native renal cells  and stimulates them to produce other cytokines, growth factors, and chemokines, such as interleukin-8 (IL-8), monocyte chemoattractant protein (MCP)-1, and macrophage-colony stimulating factor (M-CSF) [α upregulates the expression of endothelial and leukocyte adhesion molecules, which mediate the adhesion of monocytes, lymphocytes, and granulocytes to activated endothelium and their subsequent migration [α may promote renal damage in diabetic nephropathy through several mechanisms. It stimulates the production of endothelin-1 and therefore vasoconstriction and reduction of glomerular blood flow and glomerular filtration rate. It has a cytotoxic effect on glomerular cells and stimulates apoptosis. It disrupts the intracellular junctions of the glomerular filtration barrier and increases its permeability resulting in the development of albuminuria [α serves as a biomarker of renal inflammation, and urinary TNF-α and neutrophil gelatinase-associated lipocalin (NGAL) were predictors of renal impairment in diabetic patients [Human TNF-f 17 kDa . Human The urine , 29. TNF (M-CSF) . TNF-α uigration , 31. TNFuminuria . Urinarypatients .α levels, while other investigators described higher serum TNF-α levels both in macroalbuminuric and in microalbuminuric compared with normoalbuminuric patients [α levels and the latter were higher in patients with microalbuminuria than in those with normoalbuminuria. Interestingly, in the present study, multivariate analysis showed that urinary TNF-α was the only independent predictor of the degree of microalbuminuria. Recent studies have shown that TNF-R2 inflammatory pathway is predominantly involved in the progression of albuminuria during the early stages of diabetic nephropathy. Therapeutic strategies aiming for TNF receptor inhibition or TNF production showed improvement of glycemic control, reduction of albuminuria, and improvement of renal function [In our study, microalbuminuria was not significantly associated with serum TNF-patients . A signifunction –37.α levels suggesting mainly intrarenal production of this cytokine and therefore local and nonsystemic activation of the inflammatory response. The above hypothesis is supported by the lack of correlation between microalbuminuria and CRP, fibrinogen and serum TNF- α levels in our patients. However, it should be noted that it remains unclear whether TNF- α urinary excretion is a marker of glomerular permeability or reflects tubulointerstitial damage. Although the majority of the patients had normal renal function and accordingly minimal or absent tubulointerstitial changes, further studies are needed with measurements of tubulointerstitial markers such as alpha1-microgloburin and/or NGAL to identify the exact source of renal TNF-α production.Overall, the above findings suggest an important role of this cytokine in the pathogenesis and progression of diabetic nephropathy. In addition, in the present study, no correlation was observed between serum and urinary TNF-α levels and other microvascular or macrovascular diabetic complications such as diabetic neuropathy and cardiovascular disease indicating an early and important role for this cytokine in the pathogenesis of renal disease. Since the majority of the patients had normo- or microalbuminuria and well-preserved renal function, it appears reasonable to conclude that, during the early stages of diabetic nephropathy, local inflammatory pathways, associated with increased production and excretion of TNF-α, are activated and contribute to kidney damage progression. In more advanced stages, a systemic inflammatory response could be activated leading to the development of several other micro- and macrovascular complications.Finally, in our patients, no correlation was found between serum or urinary TNF-The present study has some limitations and shortcomings that should be considered. First, patients were classified based on one morning spot urine sample, with no repetitive measurements or timed collection being performed. Moreover, we did not include in our analysis patients with macroalbuminuria as our aim was to investigate initially biomarkers of inflammation during the early stages of diabetic nephropathy. However, it would be very interesting to investigate whether the correlation between TNF-alpha and ACR persists in patients with various stages of diabetic nephropathy. Finally, in the present study, no correlation was observed between eGFR and TNF-alpha levels as the majority of the enrolled patients demonstrated normal eGFR values and it would be also interesting to study the above association across a wide range of eGFR. We are currently recruiting patients with macroalbuminuria and/or renal function impairment in order to investigate the above important issues.α levels are associated with the presence and severity of microalbuminuria indicating that its intrarenal production is involved in the pathogenesis and progression of diabetic nephropathy. Further studies are needed to elucidate the exact role of this cytokine in diabetic nephropathy.In conclusion, in patients with type 2 diabetes mellitus urinary, but not serum, TNF-


Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria.In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death.P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death , comparable to LA  (combined area under the receiver operating characteristic curve 0.81).Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean±SD) was elevated in severe malaria (8.2 mEq/L±4.5) and severe sepsis (8.6 mEq/L±7.7) compared with uncomplicated malaria (6.0 mEq/L±5.1) and encephalopathy (6.6 mEq/L±4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and β-hydroxybutyric acid  contains supplementary material, which is available to authorized users. Severe falciparum malaria remains a leading cause of mortality in the tropics. The case fatality rate in adult patients remains between 15 % and 40 % in resource-constrained hospital settings, and it is 11 % in intensive care units in resource-rich settings . A betteThe degree of metabolic acidosis in severe malaria is a strong prognosticator for a fatal outcome. Acidosis is associated with hyperlactataemia, and lactic acid (LA) has therefore been considered a major contributor to hydrogen ion accumulation , 5. In cUsing the modified physicochemical approach of Stewart, the presence of an unmeasured anion load was detected in patients with severe malaria , 16. TheUsing a recently developed liquid chromatography-mass spectrometry (LC-MS) assay , we quanPlasmodium falciparum parasites visualised on a peripheral blood film, and severe malaria was distinguished from uncomplicated malaria by the presence of one or more markers of severity according to modified World Health Organisation (WHO) criteria as defined by Tran et al.  + [K+] + [Ca2+] + [Mg2+] − [Cl−] − [lactate−], with all concentrations expressed in milliequivalents per litre. The effective strong ion difference (SIDeff) was calculated by inclusion of the charges contributed by HCO3−, albumin and inorganic phosphate, using calculations described previously  2.1–7.0), compared with 4.2 % in patients with bacterial sepsis (IQR 2.0–15.6). The median proportion of SIG attributable to HPLA alone was 0.13 % in severe malaria (IQR 0.09–0.21), and 0.08 % in sepsis (IQR 0.04–0.18); these contributions were significantly different  and β-HBA . Weaker correlations were seen between bilirubin and all four acids and between the parasite biomass marker PfHRP2 and all four acids. GCS score correlated inversely with all four acids. The complete correlation matrix is provided in Additional file Plasma creatinine was correlated with HPLA . In the model of plasma acids in 138 patients (39 of whom died), HPLA and LA had independent prognostic value, with the combined model providing an AUROCC of 0.81. Among 112 participants with available urine specimens (34 of whom died), LA was the only urinary acid of significant independent prognostic value. The AUROCC for a model based on urinary LA alone was 0.72.Logistic regression models were developed for patients with severe malaria, with in-hospital death as the dependent variable, to assess the prognostic value of plasma and urine organic acid concentrations Table . An alteMetabolic acidosis with an increased SIG and high SBD is a prominent feature of severe falciparum malaria, and severity of the acidosis is closely linked to mortality , 16, 23.l-lactate isomers, whereas LC-MS detects both l- and d-isomers. Human metabolic pathways produce only l-lactate, but both isomers are produced by P. falciparum, as the parasite relies heavily on incomplete oxidation of glucose into LA for its own energy supplies [d-lactate can accumulate in the context of multiorgan failure, with increased transposition of bacteria from the ischaemic gastrointestinal tract [d-lactate can cause an encephalopathy in humans ranging from disorientation to coma [d-lactate could contribute to the pathogenesis of severe malaria requires further quantification and study.LA was detectable in plasma at a considerably higher concentrations than any other organic acid and was a strong independent predictor of death in patients with severe malaria, with a higher AUROCC than reported previously . We conssupplies . Bacterial tract –28, and  to coma . The hypl-tyrosine catabolism [Enterococcus faecalis, Escherichia coli, bifidobacteria and lactobacilli) can be a source of HPLA [HPLA was identified at higher concentrations in the plasma and urine of participants with severe malaria than in reference groups, and it was a strong and independent predictor of mortality in patients with severe malaria. Although total HPLA concentrations were relatively low, the high prognostic value of HPLA may reflect perturbations in associated metabolic pathways, with related acids also contributing to the total acid load. Alternatively, this may indicate strong biological activity of the anion at low concentrations or local accumulation in affected tissues. HPLA is a product of tabolism . Plasma tabolism , 31–35. tabolism , 36. Uritabolism . In thestabolism , 39. In  of HPLA . This co of HPLA –43. A poβ-HBA was high in plasma and urine of patients with severe malaria but highest in patients with severe sepsis. Concentrations were not independently correlated with mortality. β-HBA is synthesised in the liver from free fatty acids, and increased production of ketoacids can be related to the augmented metabolic demand, coupled with reduced oral caloric intake, in severe illness , 44, 45.Concentrations of α-HBA were high in patients with severe malaria, sepsis or encephalopathy, but they were not an independent predictor of mortality. α-HBA is a product of threonine and methionine catabolism, but it is also produced in glutathione synthesis during the conversion of cystathionine to cysteine. The glutathione pathway is upregulated to counteract oxidative stress, which is increased during severe malaria , 46. RaiWe acknowledge the limitations of this hypothesis-generating study and the need for further, targeted exploration of the mechanisms by which the identified acids influence the pathogenesis of malaria. The heterogeneity of organ dysfunction in both severe malaria and severe sepsis complicates our understanding of both conditions, particularly with regard to the metabolic pathways implicated. This heterogeneity reduces the statistical power of our analysis; yet, significant conclusions emerge.Concentrations of HPLA were significantly higher in patients with severe malaria than in patients with sepsis, febrile encephalopathy or uncomplicated malaria, which suggests a disease-specific role. HPLA also has prognostic significance independent of LA. Further research into the kinetics and biological effects of these acids, as well as into the contributing metabolic pathways, is warranted.Previously unmeasured organic acids could be identified in patients with severe falciparum malaria using a novel targeted LC-MS approach.A tyrosine metabolite and two ketoacids were detected at increased concentrations in patients with severe malaria.Lactate and 4-hydroxyphenyllactic acid were predictive of mortality in patients with severe malaria.Our findings highlight metabolic pathways potentially implicated in disease pathogenesis.


MCI (0%) and vs. AD (4.7%). Furthermore, higher education in ASYMAD vs. MCI and AD (p < 0.05) was found. These novel autopsy-verified findings support the hypothesis of the beneficial effect of APOε2 and education, both which seem to act as contributing factors in delaying or forestalling the clinical manifestations of AD despite consistent levels of AD pathology.Asymptomatic Alzheimer's disease (ASYMAD) subjects are individuals characterized by preserved cognition before death despite substantial AD pathology at autopsy. ASYMAD subjects show comparable levels of AD pathology, i.e. β-amyloid neuritic plaques (Aβ-NP) and tau-neurofibrillary tangles (NFT), to those observed in mild cognitive impairment (MCI) and some definite AD cases. Previous clinicopathologic studies on ASYMAD subjects have shown specific phenomena of hypertrophy in the cell bodies, nuclei, and nucleoli of hippocampal pyramidal neurons and other cerebral areas. Since it is well established that the allele APOε4 is a major genetic risk factor for AD, we examined whether specific alleles of APOE could be associated with the different clinical outcomes between ASYMAD and MCI subjects despite equivalent AD pathology. A total of 523 brains from the Nun Study were screened for this investigation. The results showed higher APOε2 frequency ( These older individuals with preserved cognition and consistent amounts of AD pathology at autopsy have been termed asymptomatic AD (ASYMAD) subjects. ASYMAD subjects not only have comparable levels of AD pathology to those found in mild cognitive impairment (MCI) subjects and some definite AD patients but also have an identical cerebral localization [in vivo” functional neuroimaging studies [Many decades of clinicopathologic investigations on sporadic Alzheimer's disease (AD) have enormously contributed to the definition of the main pathologic lesions associated with AD, i.e.  lesions , 2. Howelarified -5. The h decline -12. One lization , 15. Othlization  or “preclization . Those i studies -22.a priori” whether these subjects would have remained cognitively preserved or eventually would have progressed to MCI or AD had they lived longer. The term ASYMAD then has the advantage to not imply necessarily a forthcoming or future cognitive decline [The reason to term older individuals cognitively preserved but positive for AD pathology at autopsy using the acronym ASYMAD is because it is virtually impossible to know “ decline . In addi decline .β-NP and NFT staging without taking into account any clinical or dementia history of the subject that received the autopsy.It is significant to recall here that a clinicopathologic dissociation between AD pathology and cognition was already recognized a few decades ago -31 withoIn previous studies -37, a maThis new clinicopathologic investigation aimed to go beyond the morphometric characterizations , 36 and There are very few large epidemiological studies that include analyses of normal aging and AD, and an autopsy program. Among those rarer epidemiological studies, the Nun Study has been a historical one . RenderiDemographic, educational, cognitive, neuropathologic, and APOE allele distribution data across groups are summarized in Table ASYMAD and C did not differ for the last MMSE and ADLs mean scores. By contrast, AD showed significantly lower MMSE and ADLs mean scores compared to all other groups.β-NP CERAD scores showed no statistical difference between ASYMAD and MCI, whereas differences were found between ASYMAD and AD (p < 0.05); and between MCI and AD (p < 0.05). Braak-NFT scores were different across comparisons when all stages (0-VI) were considered: ASYMAD vs AD (p < 0.001), MCI vs AD (p < 0.01), and ASYMAD vs MCI (p < 0.05). However, when the analysis of Braak staging was performed including only the first five stages (0-IV), no differences were observed between ASYMAD vs. MCI and even between MCI and AD.Ap < 0.01) and AD . No different APOε2 frequency was detected between ASYMAD and C (13.6%). While no differences were observed in APOε3 frequency across all groups, lower APOε4 frequency was observed in ASYMAD (3.8%) compared to MCI  and AD . Importantly, no different APOε4 frequency was found between ASYMAD and C (0%). All comparisons for APOE allelic frequencies are shown in Figure Higher APOε2 frequency was found in ASYMAD 19.7%) compared to MCI  in PCG  compared to ICG   in ICG (ε4 = 20.6%) compared to PCG (ε4 = 2.0%).Moreover, APOε2 frequency was significantly higher (p = 0.02) group. Correlation analyses (Spearman's test) between APOε2 and the highest level of attained education (master or higher), however, did not show significant correlations across all groups, or any other possible correlation between groups comparison.The analyses show significant differences on education levels between PCG compared to ICG (The most important findings of this clinicopathologic study are: a) higher APOε2 frequency and b) higher educational levels in ASYMAD vs. MCI and AD. These findings suggest a beneficial effect of APOε2 and education on later cognition even in presence of AD pathology burdens that are equivalent to those found in the MCI and some definite AD cases. Previous epidemiological studies have reported a protective role of APOε2  and highβ and tau depositions, and are possibly linked to multiple biological mechanisms [In terms of pathogenic sequence, our findings suggest that the possible beneficial effects of APOε2 and higher education lie downstream to both Achanisms -64. A rechanisms , which achanisms  is diffechanisms  but alsochanisms  and cognchanisms , 48, 49.Moreover, experimental studies indicate that APOε2 has a protective role in both the peripheral and central nervous system , 67. FurRemarkably, APOε3 frequency was not different across all groups in our investigation. This suggests that it is actually the interplay between APOε2 and APOε4 to weigh heavily on the risk of developing MCI and AD, or remaining cognitively unscathed. Importantly, only the presence of APOε2 is insufficient to avoid the AD pathology deposition, as demonstrated by the conspicuous amount of it in ASYMAD subjects. APOε2, rather, seems to have beneficial properties in reducing the consequences of AD pathology accumulation or even in reducing it .APOE distribution in the Nun Study is similar to that of Caucasian populations . HoweverHow common is ASYMAD? In our series, out of 24 cognitively preserved subjects (C+ASYMAD) older than 85 years (mean age = 88.2±3.9), 54.0% showed severe levels of AD pathology but unimpaired cognition (ASYMAD). A previous large autopsy-cohort study  showed tβ-NP and NFT, but one brain has a higher concentration of toxic and specific soluble Aβ or tau oligomers than the other [It is not possible to transfer our findings to the general population without caveats. We cannot exclude that the higher educational levels of the Nun Study participants contributed to a higher “cognitive reserve” . In the he other , and itshe other .Subjects were identified by querying the database of the Nun Study, which includes clinical and cognitive data, educational information, neuropathologic evaluations with definite diagnoses, and APOE genotypes. Participants in the Nun Study are Catholic sisters belonging to the School Sisters of Notre Dame congregation living in various communities in the Midwestern, eastern, and southern regions of the US . At the In the present investigation only the participants that underwent autopsy were considered. The total sample size of the cohort with brain autopsy available was 523, that is, the 77.1% of the entire eligible cohort. The methods used to assess the cognitive status have been described in previous publications . BrieflyIntact (Preserved) Cognition: intact scores on all cognitive tests and activities of daily living (ADL). This category defined the cognition level of both age-matched controls (C) and ASYMAD subjects.Mildly Impaired Cognition (with memory impairment): intact on MMSE and ADL, but impaired on the Delayed Word Recall and one or more of the other three cognitive tests. This category is equivalent to mild cognitive impairment (MCI) subjects based on Mayo Clinic and AA-NIA MCI criteria , 54.Demented: met clinical criteria for dementia as described above.β-NP was assigned by semi-quantitative and age-adjusted scores  according to CERAD pathologic criteria [Subjects with complete physical, neurologic, and cognitive assessment in an interval prior to death  of ≤1.0 year.Neuropathologic assessments including CERAD pathologic diagnostic and Braak-NFT scoresAvailability of APOE genotype.The Nun Study had neuropathological data on a total of 523 autopsy brains. These brains were removed and examined macroscopically after fixation in 10% buffered formaldehyde for at least 2 weeks. Tissues blocks were dissected from middle frontal gyrus, superior and middle temporal gyri, inferior parietal lobule, occipital cortex (area 17-18), entorhinal cortex, hippocampus, amygdala, thalamus, basal ganglia and cerebellum. Tissue blocks were processed and embedded in paraffin, cut at 10 μm, and stained with hematoxylin-eosin (H&E) and Hirano-silver method. The severity of Acriteria , and NFTcriteria . Out of Met the criteria for mixed dementia. This exclusion criteria was particularly important to minimize the influence of cerebro-vascular pathologies on the cognitive deficits.Met the criteria for dementia with Lewy bodies, Parkinson's disease with dementia, Pick's disease, Creutzfeldt-Jacob disease.Showed hippocampal sclerosis, tauopathies, or primary or secondary brain tumors.Subjects were excluded if:n = 11)Age-matched controls (C): subjects with intact cognition and no significant AD pathology at autopsy  (n = 13)Asymptomatic AD (ASYMAD): subjects with intact cognition and AD pathology at autopsy  (n = 15)Mild cognitive impairment (MCI): subjects with MCI diagnosis and AD pathology at autopsy  (n = 116).Alzheimer's disease (AD), patients with clinical dementia and diagnosis of definite AD at autopsy   or from brain tissues as previously described .ANOVA was used to compare continuous variables  across all groups; while Fisher's exact test was used to compare categorical variables .For APOE allelic frequency analyses, we used Fisher's exact test for a 3-by-4 contingency table comparing the frequencies of all three APOE allele across all groups. Then, we carried out analyses to examine the hypothesis that APOε2 was enriched in ASYMAD by conducting additional Fisher's exact test to compare the frequencies of APOε2 vs. all other alleles in ASYMAD vs MCI, AD, and C, separately.In addition, an exact Fisher's test was performed to compare the APOE allelic frequencies in two larger combined groups: 1) subjects with cognitive deficits (MCI+AD) ; and 2) subjects without cognitive deficits (C+ASYMAD) . This analysis was performed to investigate possible effects of APOε2 exclusively based on the documented cognitive assessment shortly before death. Separate analyses using an exact Fisher's test were performed to compare ICG and PCG on the highest educational level attained. Finally Spearman's test for nonparametric data (APOε2 frequencies) and highest level of education attained (master or higher) across all groups, expressed as percentages, was also performed to test if significant correlation between APOε2 and higher education was possibly present.