Amosy E. M'Koma

Proteomic profiling of mucosal and submucosal colonic tissues yields protein signatures that differentiate the inflammatory colitides

Background: Differentiating ulcerative colitis (UC) from Crohns colitis (CC) can be difficult and may lead to inaccurate diagnoses in up to 30% of inflammatory bowel disease (IBD) patients. Much of the diagnostic uncertainty arises from the overlap of clinical and histologic features. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) permits a histology‐directed cellular protein analysis of tissues. As a pilot study, we evaluated the ability of histology‐directed MALDI‐MS to determine the proteomic patterns for potential differences between CC and UC specimens. Methods: Mucosal and submucosal layers of CC and UC colon resection samples were analyzed after histologic assessment. To determine whether MALDI‐MS would distinguish inflammation, the uninflamed (n = 21) versus inflamed submucosa (n = 22) were compared in UC and the uninflamed (n = 17) versus inflamed submucosa (n = 20) in CC. To determine whether there were proteomic differences between the colitides, the uninflamed UC submucosa (n = 21) was compared versus the uninflamed CC submucosa (n = 17), the inflamed UC submucosa (n = 22) was compared versus the inflamed CC submucosa (n = 20), and inflamed UC mucosa versus inflamed CC mucosa. Pairwise statistics comparisons of the subsets were performed. Results: Pairwise comparative analyses of the clinical groups allowed identifying subsets of features important for classification. Comparison of inflamed versus uninflamed CC submucosa showed two significant peaks: m/z 6445 (P = 0.0003) and 12692 (P = 0.003). In the case of inflamed versus uninflamed UC submucosa, several significant differentiating peaks were found, but classification was worse. Comparisons of the proteomic spectra of inflamed submucosa between UC and CC identified two discrete significant peaks: m/z 8773 (P = 0.006) and 9245 (P = 0.0009). Comparisons of the proteomic spectra of uninflamed submucosa between UC and CC identified three discrete significant peaks: m/z 2778 (P = 0.005), 9232 (P = 0.005), and 9519 (P = 0.005). No significantly different features were found between UC and CC inflamed mucosa. Conclusions: MALDI‐MS was able to distinguish CC and UC specimens while profiling the colonic submucosa. Further analyses and protein identification of the differential protein peaks may aid in accurately diagnosing IBD and developing appropriate personalized therapies. (Inflamm Bowel Dis 2011;)

Chemokine markers predict biochemical recurrence of prostate cancer following prostatectomy

Purpose: Stratifying patients who have a high risk of prostate cancer recurrence following prostatectomy can potentiate the use of adjuvant therapy at an early stage. Inflammation has emerged as a mediator of prostate cancer metastatic progression. We hypothesized that chemokines can be biomarkers for distinguishing patients with high risk for biochemical recurrence of prostate cancer. Experimental Design: In a nested case-control study, 82 subjects developed biochemical recurrence within 5 years of prostatectomy. Prostate tissues from 98 age-matched subjects who were recurrence-free following prostatectomy in the same period were the controls. A high-throughput lectin-based enrichment of prostate tissue enabled multiplex ELISA to identify the expression of three chemokines to discriminate the two patient populations. Results: The expression of CX3CL1 and IL-15 in prostate tissue was associated with 5-year biochemical recurrence-free survival following prostatectomy. However, the expression of chemokine ligand 4 (CCL4) was associated with biochemical recurrence. Multivariable logistic regression model combining preoperative prostate-specific antigen, Gleason score, surgical margin, and seminal vesicle status with the three chemokines doubled the specificity of prediction at 90% sensitivity compared with use of the clinicopathologic variables alone (P < 0.0001). Survival analysis yielded a nomogram that supported the use of CX3CL1, IL-15, and CCL4 in predicting 1-, 3-, and 5-year recurrence-free survival after prostatectomy. Conclusions: Each of the three chemokines can serve as independent predictors of biochemical recurrence. However, the combination of chemokine biomarkers plus clinicopathologic variables discriminated prostatectomy subjects for the probability of biochemical recurrence significantly better than clinicopathologic variables alone.

Follow-up results of hematology data before and after restorative proctocolectomy

PURPOSE: This study was designed to investigate the development of anemia during functional ileoanal pouch. METHODS: Eighty-three patients received an ileoanal pouch between 1980 and 1987. The hematology data deviations among the preoperative period, defunctionalized stage, and after ileal pouch in function were monitored. Aspects studied included serum hemoglobin, iron, folates, vitamin B12, white blood cell, erythrocyte sedimentation rate, pt-B12, and fat absorption. Specimens were collected before colectomy, during ileostomy prior to pouch operation, before loop ileostomy closure, and at 6, 12, 18, 24, and 36 months after loop ileostomy closure. RESULTS: Two patients developed iron deficiency anemia after 2.5 and 5 years of pouch function: one patient, a vegetarian, had low hemoglobin, iron, and vitamin B12 postoperatively, the other patient developed low iron and hemoglobin 5 years after the operation. Significant elevations of serum hemoglobin, iron, and folates were seen preoperativelyvs postoperatively from 12346±2.845 g/l, 10.282±0.992 μmol/l, and 9.983 ±1.308 mmol/l to 138.842±1.563 g/l (P<0.0001), 17.544±1.529 μmol/l (P<0.0003), and 16.784±1.757 mmol/l (P<0.01) (mean±SE) of the defunctionalized loop ileostomy. Serum B12 decreased insignificantly. After loop ileostomy closure, at 6 and 36 months of functional ileal pouch-anal anastomosis, the elevations were still siginificant; serum hemoglobin wasP<0.0001 andP<0.01, and serum iron wasP<0.001 andP< 0.01, respectively. Vitamin B12 levels decreased insignificantly at six months in controls and significantly (P< 0.01) at 36 months. There was a significant increase of serum folates (P<0.01 andP<0.001). Patients with low iron were 50 percent at precolectomy, 23 percent with ileostomy, 16 percent with loop ileostomy, 15 percent at six-month follow-up, and 11 percent at 12-month follow-up. Although only 3 percent and 11 percent of the patients with ileal pouch-anal anastomosis had low serum vitamin B12 values at the 12-month and 36-month follow-up, 31 percent and 36 percent had decreased Schilling tests. Thirty-three percent and 41 percent had decreased14C-triolein breath tests. In five patients vitamin B12 deficiency began during the first six months of pouch function: in two patients after one year and in one patient after two years. Eight of 83 patients have had substitution therapy with vitamin B12. The therapy was discontinued in three patients after two to four years; these patients developed no further symptoms and had normal Schilling tests in the succeeding 30 months to 46 months. Five patients continue with substitution after 40 months to 68 months. During the functional period, serum erythrocyte sedimentation rate and white blood cells were elevated in some controls on different occasions. Folates were normal throughout the functional period. CONCLUSION: For predicting hematologic data outcome in patients with functional ileal reservoir, the results justify the necessity for control during both manipulative and functional periods until evaluations are reliable and satisfying.

Proteomic patterns of colonic mucosal tissues delineate Crohn's colitis and ulcerative colitis

Although Crohns colitis (CC) and ulcerative colitis (UC) share several clinical features, they have different causes, mechanisms of tissue damage, and treatment options. Therefore, the accurate diagnosis is of paramount importance in terms of medical care. The distinction between CC/UC is made on the basis of clinical, radiologic, endoscopic, and pathologic interpretations but cannot be differentiated in up to 15% of inflammatory bowel disease patients. Correct management of this “indeterminate colitis” depends on the accuracy of future, and yet not known, destination diagnosis (CC/UC).

Evolution of the restorative proctocolectomy and its effects on gastrointestinal hormones

Gastrointestinal (GI) peptide hormones are chemical messengers that regulate secretory, mechanical, metabolic, and trophic functions of the gut. Restorative proctocolectomy (RPC) or resection of the colon and rectum with maintenance of intestinal continuity through the construction of an ileal pouch reservoir and preservation of the anal sphincters has become the standard of care for the surgical treatment of ulcerative colitis and familial adenomatous polyposis. The manipulation of the digestive system to create the ileal pouch involves altering gut-associated lymphoid tissue among other anatomic changes that lead to changes in GI peptides. In addition, the ileal pouch epithelium responds to a wide variety of stimuli by adjusting its cellularity and function. These adaptive mechanisms involve systemic factors, such as humoral and neural stimuli, as well as local factors, such as changes in intestinal peristalsis and intraluminal nutrients. There have been conflicting reports as to whether the alterations in GI hormones after RPC have actual clinical implications. What the studies on alterations of GI peptides’ response and behavior after RPC have contributed, however, is a window into the possible etiology of complications after pouch surgery, such as pouchitis and malabsorption. Given the possibility of pharmacologically modifying GI peptides or select components of adaptation as a therapeutic strategy for patients with ileal pouch dysfunction or pouchitis, a clear understanding of human pouch mucosal adaptation is of paramount importance. In this review, we summarize the evolution of the RPC and its effects on the GI hormones as well as their possible clinical implications.

Effect of restorative proctocolectomy on gastric acid secretion and serum gastrin levels: a prospective study.

PURPOSE: The aim of the present study was to analyze gastric acid secretion after restorative proctocolectomy, because it has been shown that ileal resection or exclusion may increase gastric acid secretion. An increased output of gastric acids may decrease the intestinal passage time and contribute to looser stools. METHODS: Eleven patients who had elective colectomy and ileoanal pouch because of ulcerative colitis were investigated. Eight patient were males. Eight S-pouches and three J-pouches were constructed. Gastric acid secretion (retention, basic, and stimulated) was studied, together with serum gastrin, pentagastrin, and pepsinogen, in patients before colectomy and after having had the pelvic pouch functioning for 12 months. RESULTS: A significant increase, compared with preoperative levels, in retention, basic, and stimulated gastric acid secretion was found after 12 months with the pouch functioning. Levels of serum gastrin, pentagastrin, and pepsinogen were unchanged. CONCLUSION: Restorative proctocolectomy leads to a significant increase in gastric acid secretion. These findings may be of importance with regard to intestinal passage time and consistency of the stools.

Diagnosis of inflammatory bowel disease: Potential role of molecular biometrics

Accurate diagnosis of predominantly colonic inflammatory bowel disease (IBD) is not possible in 30% of patients. For decades, scientists have worked to find a solution to improve diagnostic accuracy for IBD, encompassing Crohns colitis and ulcerative colitis. Evaluating protein patterns in surgical pathology colectomy specimens of colonic mucosal and submucosal compartments, individually, has potential for diagnostic medicine by identifying integrally independent, phenotype-specific cellular and molecular characteristics. Mass spectrometry (MS) and imaging (I) MS are analytical technologies that directly measure molecular species in clinical specimens, contributing to the in-depth understanding of biological molecules. The biometric-system complexity and functional diversity is well suited to proteomic and diagnostic studies. The direct analysis of cells and tissues by Matrix-Assisted-Laser Desorption/Ionization (MALDI) MS/IMS has relevant medical diagnostic potential. MALDI-MS/IMS detection generates molecular signatures obtained from specific cell types within tissue sections. Herein discussed is a perspective on the use of MALDI-MS/IMS and bioinformatics technologies for detection of molecular-biometric patterns and identification of differentiating proteins. I also discuss a perspective on the global challenge of transferring technologies to clinical laboratories dealing with IBD issues. The significance of serologic-immunometric advances is also discussed.

Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

Inability to distinguish Crohns colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohns colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohns colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohns colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohns disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohns colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohns colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohns colitis.

Advances and challenges in the diagnostic accuracy for surgical management of inflammatory bowel disease: Potential role of molecular biometrics

Innate immune system is a universal form of host defense against infections. The recognition of the innate immunity is based on a limited number of encoded receptors that have evolved to recognize microbial metabolism products. The recognition of these molecular structures allows the immune system to distinguish its own infectious components from non-communicable structures. The immune suppression is a hallmark of sepsis. The complement system is activated in the early stages of sepsis, generating large amounts of anaphylatoxin C5a. Complement and TLRs (toll-like receptors) family are two major upstream sensors and effectors systems of innate immunity. It was found that TLR4 and complement system are involved in the initiation of the inflammatory response in sepsis. Clinical studies in which TLR4 was blocked have not shown beneficial effects. TLRs, that are a subfamily of PRRs (pattern recognition receptors), have emerged as the crucial receptors for the recognition of DAMPs (Damage-associated molecular pattern molecules). Recently, a special form of non-coding genetic material called microRNA has been highlighted in the complex cascade of sepsis. The individual role of every microRNA and the exact role of microRNA network are under investigation. Currently, studies are performed in order to find micro RNA to be used as biomarkers of sepsis. Researches are performed to determine microRNA, small fragments of non-coding RNA, in order to distinguish between patients with sepsis and healthy patients, and if the plasma levels of microRNA correlate with the severity of the disease. Recent researches report that the regulation of gene expression through microRNA plays a very important role in the following cellular processes, for example: apoptosis, the differentiation process, and the cell cycle.Background: Vitamin D is a pivotal factor not only in disorders that involve calcium metabolism, such as osteoporosis and osteomalacia as well as an immunomodulatory effect as noted in several autoimmune conditions in diseases. Very low levels of vitamin D were also demonstrated in systemic sclerosis (SSC) patients. Patients with vitamin D deficiency showed longer and more severe disease. Furthermore, an inverse relationship was found between skin involvement and vitamin D serum concentrations. Associations were found Between Systemic Sclerosis pattern of disease and Scleroderma-Specific Autoantibodies. Novel research demonstrated the presence and importance of anti-vitamin D antibodies in SLE; this motivated our research team to seek for similar antibodies among scleroderma patients.T role of intracellular recognition molecules like NODs expressed by cells of the female reproductive tract (e.g. Fallopian tube epithelium) in the response to chlamydial infection, or any STI, remains poorly understood. A suboptimal innate immune response may result in a permissive environment for pathogen colonization, whereas an over-exuberant response will cause excessive inflammation and tissue damage. Modulation of the host response to infection is an attractive alternative or adjuvant approach to antibiotic therapies in treatment of genital tract infections. Genome sequence analysis has revealed that Chlamydia possesses numerous novel genes that might be involved in the manipulation of the host cells. The infected cells often display altered metabolic, immunological and cell biological characteristics, however, at the same time the microbes have to maintain the integrity and viability of host cells before completing their own intracellular replication. To achieve this goal chlamydiae have evolved the ability to both prevent the infected cells from undergoing apoptosis induced by intracellular stress and to protect these cells from recognition and attack by lymphocytes. Analysis of C. trachomatis genome had identified more than two dozens of open reading frames encoding proteins with potential proteolytic activity. Some of these proteases may be used to target host cell proteins because some proteins are cleaved and/or degraded in infected cells. The attacked host proteins include transcriptional factors, pro and anti apoptoytic proteins, DNA repairing enzymes, cyclins and cytoskeletal protein. However, survival strategies of Chlamydia at the tissue level and the relevance of these findings in disease pathogenesis have yet to be determined. Since till date a Chlamydia vaccine is unavailable, a better definition of human immune response along with Chlamydial survival strategies needs to remain an important research priority if we are to develop a vaccine against C. trachomatis infection that has protective and not deleterious effects.I of angiogenesis is currently perceived as one of the promising strategies in the treatment of cancer. The antiangiogenetic property of thalidomide has inspired a second wave of research on this teratogenic drug. The aim of the current study is to investigate the anti-proliferative and the anti-angiogenic activities of two thalidomide dithiocarbamate analogues by determining their anti-proliferative abilities towards human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 human breast cancer cell lines. Furthermore, their effects on the expression level of TNF-α and VEGF165 mRNA in MDAMB-231 had been assessed. Moreover, we evaluated their effects on angiogenesis using HUVECs through tube formation assay and wound healing assay. Results illustrated that, MDA-MB-231 and HUVECs proliferations were not significantly affected by thalidomide at 6.25-100 μM. Besides, thalidomide failed to block angiogenesis at similar concentrations. However, thalidomide diminished the expression level of TNF-α and VEGF165 by 27% and 34.4%, respectively. Conversely, thalidomide dithiocarbamate analogues 1 and 2 were significantly established anti-proliferative action in MDA-MB-231 and HUVECs without causing cytotoxicity. Two analogues showing powerful anti-angiogenicity in the tube formation assay and wound healing assay as well as analogue 1 demonstrated more potent inhibition ability to suppress TNF-α (79%) and VEGF165 (52%) than analogue 2, TNF-α (56%) and VEGF165 (41%). Analogue 1 consistently showed the highest potency and efficacy in all assays. Taken together, our results support the further development and evaluation of novel thalidomide dithiocarbamate analogues as anti-tumor and anti-angiogenic agents.Background: Acinetobacter baumannii is an important opportunistic pathogen which causes complications in hospitalized patients, especially those in ICU. The aim of this study was to determine the frequency of class 1 and 2 integrons in multi-drug resistance A. baumannii and to investigate the association between the presence of integrons and antibiotic resistance patterns. Methods: A total of 40 A. baumannii strains were isolated from 372 ICU patients from June to Oct 2012. A. baumannii was detected in 50% of tracheal cultures, 15% in blood, 15% in urine samples, and 22.5% in other locations. In accordance with CLSI 2011, 12 antibiotics were used through disc diffusion method. Existence of integron classes was investigated by PCR assay with the amplification of integrase genes. Results: The most effective antibiotic against Acinetobacter baumannii was polymyxin B with 100% susceptibility, followed by meropenem, piperacillin, cotrimoxazole, ceftazidime with 100% resistance; this was followed by ciprofloxacin 97.5%, tetracycline, 92.5%, imipenem 62.5%, and gentamicin 60% resistance. The presence of integron class 1 was 7.5%, class 2 was 67.5%, and non-integron was 20%. Conclusion: The association between multidrug resistance and class 2 integron was not statistically significant. Other factors accounting for the lack of significance of the findings may be the impact of other resistance determinants such as transposons or plasmids, not investigated in the current study. Considering the increasing trend of MDR infections among ICU patients with critical problems in follow up, the use of appropriate infection control strategy and a regular surveillance system is necessary in our hospital.C (CBBN) is a RING-domain containing protein that likely functions as a substrate receptor for the DDB1/Cul4/ CRBN E3 ubiquitin ligase (UbL) complex. Recently, CRBN was shown to be a target of the immunomodulatory (IMiD) drug thalidomide. Although known for inducing severe teratogenicity in the 1950s, this drug class is now used extensively for anticancer immunotherapy. CRBN is expressed in the hematopoietic compartment, but currently has no known function in immune regulation. Ubiquitin ligases including C-Cbl, cbl-b, GRAIL, and ITCH underlie T-cell homeostatic regulation and protect against autoimmunity. To understand the role of CRBN in T-cell function, we studied a mouse with a germline deletion of exon 3 and 4 of this gene. First, crbn-/mice are viable, fertile and normal in appearance without limb malformations. In the T-cell compartment, splenic and peripheral blood lymphocytes are increased at 3 months of age. The expanded population of splenic T-cells was also evident by immunohistochemical staining. Mature differentiated cell lineages such as CD4 and CD8 single positive (SP) thymocytes, SP peripheral T-cells and naive and memory T-cells are similar to wild-type littermates with no apparent spontaneous autoimmune features at 3 months. Similar to IMiD responses, the mature T-cells from crbn-/mice showed superior activation after T-cell receptor (TCR) stimulation. Proximal TCR signaling events including pZAP70 and pLck, cytokine production and survival are increased in knockout mice relative to wild-type littermates. In summary, our findings demonstrate a novel role for CRBN, the molecular target of thalidomide and other members of the IMiD-family, in T-cell activation.Results: A gender ratio of 3.8:1, average age onset of 27±7.3, and a mean diagnostic delay of 8 years was observed. The incidence of juvenile-onset cases was 11%. Positive family history was higher than that observed in most other countries. Both gender groups were similar in terms of ethnicity, positive family history, juvenile-onset AS, and diagnostic delay. Enthesitis was present in more than two-thirds of patients with females demonstrating a higher rate (p<0.05). Uveitis was the leading extra-articular manifestation. Overall HLA-B27 positivity was 73% and four HLA-B27 subtypes were found. HLA-B27 positivity was higher among males (78.3% vs. 55.2%; p<0.001). Disease activity was high and the functional status was poor as indicated by mean Bath AS disease activity, functional, and metrology indices. Female disease was at least as severe as male disease and more severe impairment was present in some aspects. Extra-articular manifestations and treatments modalities presented similar frequencies among genders. Biological medication was utilized less frequently than disease modifying anti-rheumatic drugs and corticosteroids. Quality of life was considerably impaired.N is the pathophysiological basis for permanent neurological disabilities in multiple sclerosis (MS); thus neuroprotection is emerging as a therapeutic approach in MS research. Modulation of excitotoxicity by inhibition of NMDARs has been suggested for neuroprotection. Decrease in Calcium-dependent protease activity secondary to NMDAR inhibition, has been postulated as a mechanism for this approach. However, this probable mechanism remains to be proven yet. Moreover, selective antagonisation of NR2B subtype of these receptors, an NMDAR subtype believed to play a more pivotal role in neurodegeneration, has not been studied too. In this study, the effect of inhibition of NR2B-containing NMDAR was evaluated on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE induction was done using MOG in C57/Bl6 mice. Therapeutic administration of different doses of highly selective NR2B-containing NMDAR inhibitor (RO25-6981) was compared with memantine (non-selective NMDAR antagonist) and vehicle in different experimental groups. Behavioral, histological and western blot analysis of Calpain and Caspase 3-dependent α-spectrin break down were studied comparatively among groups. Neurological deficits in EAE animals were more efficiently decreased by selective inhibition of NR2B-containing NMDARs. Histological studies of the spinal cords also showed decreased inflammation, myelin degradation, neuro-axonal degeneration and Calpain activity when RO25-6981was administered with higher doses. Regarding the role of NR2B-containing NMDARs in excitotoxicity, selective inhibition of these receptor subtypes appears to modulate the neurological disabilities and pathological changes in EAE. Decreasing the activity of Calcium-dependent protease, Calpain, by blockade of NR2B-containing NMDAR using high dose of RO25-6981 can be seen simultaneously. More experimental studies could be performed to suggest NR2B-containing NMDAR inhibition as a potentially effective treatment strategy for slowing down the clinical deterioration of disability in MS.Background: Information on the pattern of drug resistant tuberculosis (TB) among re-treatment cases is crucial to develop appropriate control strategies. Therefore, we conducted this study to assess the drug resistance pattern of M. tuberculosis complex (MTBC) isolates and associated factors among re-treatment cases in Jimma area, Southwest Ethiopia. Methods: Health facility-based cross-sectional study was conducted between March 2012 and April 2013 in Jimma area, Southwest Ethiopia. We included 79 re-treatment cases selected conveniently. Socio demographic and clinical data were collected using structured questionnaire. Sputum sample processing, mycobacterial culture, isolation and drug susceptibility testing (DST) were done at Mycobacteriology Research Centre (MRC) of Jimma University. All data were registered and entered in to SPSS version 20. Crude odds ratio (COR) and adjusted odds ratios (AOR) were calculated. P-values less than 0.05 were considered statistically significant. Results: Seventy-nine re-treatment cases included in the study; 48 (60.8 %) were males. Forty- seven (59.5 %) study participants were from rural area with the mean age of 31.67 ± 10.02 SD. DST results were available for 70 MTBC isolates. Majority (58.6 % (41/70)) isolates were resistant to at least one of the four first line drugs. The prevalence of multidrug-resistant TB (MDR-TB) was 31.4 % (22/70). Place of residence (AOR = 3.44 (95 % CI: 1.12, 10.60), duration of illness (AOR = 3.00 (95 % CI: 1.17, 10.69) and frequency of prior TB therapy (AOR = 2.99, (95 % CI: 1.01, 8.86) were significant factors for any drug resistance. Moreover, history of treatment failure was found to be associated with MDR-TB (AOR = 3.43 (95 % CI: 1.14, 10.28). Conclusion: The overall prevalence of MDR-TB among re-treatment cases around Jimma was high. The rate of MDR-TB was higher in patients with the history of anti-TB treatment failure. Timely identification and referral of patients with the history of treatment failure for culture and DST need to be strengthened.C T cells have the competence to attack cancer cells and eradicate a complete tumor. However, this mechanism has showed challenging for two motives. First, T cells and other elements of the immune system ignore self-molecules and cells. Second, tumor microenvironment has immunosuppressive elements capable of producing a mechanism called immunological tolerance. CD4+ T cells generate signaling molecules and activate immune cells that deliver efficient CD8+ T cell response. CD70 is essential for dendritic cells-facilitated delay of T cell tolerance initiation. CD80 and CD86 cells are implicated in the refunctionalizing the tolerized T cell. Here, these cells (CD4+, CD8+, CD70, CD80 and CD86) were obtained from a tumor prostate tissue treated with androgen ablation and were applied in a mouse model of human prostate cancer. Count cell number and purity for the particular cell population was realized by flow cytometric analysis. Preliminary results report that the activity of the CD8+T cells persisted for up to 45 days after treatment with CD4, CD70, CD80 and CD86 cells, and resulted in a significant diminution of tumor size. The stimulation of T cell infiltration and other immune cells in cancer tissues could have effects for the immunotherapeutic treatment of other hormone-related malignant tumors.

Abstract B68: Colorectal Cancer in the Setting of Inflammatory Bowel Disease: Role of Hemoglobin

Colorectal carcinoma (CRC) is a serious complication of inflammatory bowel disease (IBD) and accounts for approximately 15% of all IBD-associated deaths. The likelihood of IBD-related carcinoma is greater than that of sporadic CRC. Over one half are diagnosed at stage III or IV when cancer cells have already invaded surrounding tissues and most conventional therapeutics are limited in their success. Therefore the early detection of cancer, which is difficult in IBD, is crucial for its ultimate control and prevention. While mining the colonic mucosal and submucosal layers for biomarkers that differentiate ulcerative colitis (UC) from Crohns colitis (CC) using Matrix-assisted laser desorption/ionization (MALDI) profiling, we found a signal at m/z 5045 to be more intense in UC. Liquid chromatography–mass spectrometry (LC-MS/MS) analysis allowed identification of this signal as triply charged hemoglobin alpha chain. Macrophages are highly versatile phagocytes active in multiple roles in the immune system and key players in the inflammatory response. Their presence within the inflammatory microenvironment, in some cases, has been proven to increase transformation, angiogenesis, and immunosuppression. In hemorrhagic situations (as in UC), macrophages engulf erythrocytes that are outside the vascular bed and as a result free hemoglobin is released. Hemoglobin induces DNA damage in human colonic cells and is genotoxic. The potential carcinogenic effects of hemoglobin were documented when it was shown that hemoglobin increases the number of aberrant crypt foci in colon mucosa. In the colon, free hemoglobin is expected to increase the production of reactive oxygen (O2) species (ROS) from peroxides via the Fenton reaction, which may be the cause of cellular toxicity and eventually pro-mutagenic lesions. Intracellular reactions with active O2 can result in the initiation and progression of carcinogenesis by induction of gene mutations, chromosomal damage and cytotoxic effects. We hypothesize that elevated expression of mucosal free hemoglobin would be associated with an increased risk of CRC. To validate this hypothesis will require investigating whether hemoglobin could be classified as a proliferative or transforming agent for colon cancer cells by causing reactive oxygen species release and subsequent DNA damage. For this purpose, we will assess the cellular viability of normal colonic cell-lines, NCM 356 and NCM 460. These cell-lines will be treated with hemoglobin at different concentrations to determine the changes in levels of ROS. ROS production will be measured using C-400 staining assay and further analysis will be carried out using FACS. Additionally, we will also examine the potential cytotoxicity of hemoglobin. Supported: MMC-VICC Cancer Partnership Grant#: 3U54CA091408-09S 1; MeTRC grant#: 5U54RR026140-03, and Vanderbilt SPORE in GI Cancer Grant#: P50CA095103.