4^th International Conference and Exhibition on Immunology September 28-30, 2015 Houston, Texas, USA

Works as Professor Medical Microbiology and Immunology in Alexandria Faculty of Medicine, Egypt since 2007. Married with 3 sons and 2 daughters. Trained in Immunopathology department in NAMRU-3 in Cairo in 1991-1994 in cellular and molecular immunology. Supervised 32 theses in Medical Microbiology and Immunology. Attended 45 International Medical Conferences. Defended 20 theses. Created advanced immunity electronic learning courses for master and doctorate degrees in Medical Microbiology and Immunology. Deputy Chief editor of International Journal of Medicine and Medical Sciences. Volunteer on line Reviewer In International Research Journal of Microbiology. Member in the editorial board team of International Journal of Microbiology and Immunology Research. Coordinator of Integrated Medical Microbiology &Immunology Program for 2nd, 3rd and 4th undergraduate Medical Students. Member in New York Academia of Science, Coordinator of International Education Program of second year medical students. Member of assessor team in Permanent Committee for Promotion of Professors and Associate Professors in Medical Microbiology and Immunology in Egypt and Arab countries like Saudi Arabia, Iraq and Libya. Academic Advisor of Master degree in Medical Microbiology & Immunology in Alexandria Faculty of medicine. Member of assessor team committee in Legibility Exam. For Doctorate degrees in Medical Microbiology and Immunology in Alexandria Faculty of Medicine and Medical Research Institute, Egypt.

The highest prevalence of Hepatitis C virus (HCV) and Helicobacter pylori has been reported in Egypt but few researches among leukemic and malignant lymphoma cases. So the present study was designed to screen acute leukemia and malignant lymphoma patients for Helicobactor pylori antibodies to six virulent antigens by a new line immunoassay and correlate the clinical status to epidemiological markers of bacterial virulence factors as well as screening the same cases to HCV antibodies as a risk cofactor and correlate between both. Cases of the present study were selected from haematology unit of Alexandria Main University Hospital from June 2012 till September 2013. One hundred leukemia/ malignant lymphoma cases who were fully investigated by clinical and laboratory tests were the candidate of our screening serological study. Acute leukemia cases were early diagnosed by CBC and bone marrow examination. Malignant lymphoma was also early diagnosed by lymph node biopsy. Screening for H. pylori antibodies to six virulent antigens were performed by line immunoassay following the manufacturer’s instructions. It was LINE test kit for the quantitative detection of H. pylori specific IgG respectively IgA antibodies in human serum. Screening for HCV antibodies were done by rapid commercial test following the manufacturer’s instructions. Our results revealed that antibodies to cytotoxin associated gene (Cag A) were predominant among cases and a significant association (p<0.05) between antibodies to vacuolating (vac) antigens of Helicobacter pylori and HCV antibodies among our leukemic and malignant lymphoma cases. We can conclude that rapid non-invasive methods by line immunoassays could easily screen hematogenous malignancies to these carcinogenic pathogens (HCV and H. pylori).

Nageen Hussain has completed her PhD from University of the Punjab, Lahore-Pakistan. She is working as an Assistant Professor in the MMG department, University of the Punjab, Lahore-Pakistan. She has published more than 19 papers in reputed journals and has been serving as an Editorial Board Member of reputed journals. In 2014, she got the best poster award two times. She has organized number of workshops and conferences. Her field of interest is immunogenetics.

Systemic lupus erythematosus is an autoimmune disease which is characterized by the production of multiple antibodies. In SLE, autoantibodies react with self antigens to form immune complexes that get deposited on the tissue, resulting in inflammation and ulitmately bring about the symptoms of lupus. DNASE I gene mutations are found to be associated with SLE patients in different populations but this is the first study to be carried out on Pakistani SLE patients. A total of forty-two Pakistani SLE patients were included in this study, all fulfilling the ACR criteria. Age and sex matched healthy controls were also included. The first step was blood sample collection which was followed by DNA isolation. Later DNA was amplified by step down PCR technique to make the results reliable and thus got the band of interest that was of 165 bp. Amplified product was extracted and sequenced by Sanger dideoxy method. Bioinformatics was applied and multiple mutations were observed in DNASE I gene Exon 9.

I am Madhuri Padala presently pursuing my PhD in Biochemistry under the supervision of Dr Sritharan, Head , Molecular Diagnostics and markers , Global hospitals, Hyderabad, in the area of sepsis , inflammation and proinflammatory cytokines in collaboration with Prof.Surya S.Singh, Dept. of Biochemistry, Osmania University, Hyderabad. I have completed my masters in biochemistry from Nagarjuna University, Guntur, Andhra Pradesh and M.Phil in Biotechnology from Bharathidasan University, Trichy, Tamilnadu. My area of interests are immunology and molecular biology. Iam a recipient of all round proficiency award in BSc. My interests are reading and travelling.

Sepsis is an inherent fatal clinical syndrome and no effective therapy is actually known. In case of gram negative bacteria, lipopolysaccharides cause infection which is mediated by pro inflammatory cytokines (TNF and IL-1) and late (HMGB1). During the early stages of sepsis; the complement system is systemically activated, generating large amounts of the anaphylatoxin. C5a, which is a central molecule in the immunopathogenesis of sepsis, exerts its effects through interactions with its two C5a receptor (C5AR) and C5a-like receptor 2 (C5L2). The expression of these receptors is upregulated during sepsis, and their interactions with C5a contribute synergistically to harmful events in sepsis. We hypothesize that it’s more beneficial to block the infectious agent in the first step enabling better control on the immune system activation. Lipopolysaccharide (LPS) is the bioactive molecule present on the outer membrane of the Gram-negative bacteria. LPS interacts with sensors on the host cell membrane to activate the immune response. Thus, developing bio-molecules (peptides) which are capable of interfering with LPS at high affinity, especially to the lipid A moiety is an efficient way to neutralize the LPS toxicity. Alternatively, by blocking LPS and its serum binding protein one can block LPS toxicity. We designed based on bioinformatics data two peptides and demonstrated in vivo that both these peptides effectively blocked LPS mediated activation of cytokine production in cells. These peptides can be an effective treatment to neutralize the LPS so that sepsis can be controlled.

Victor Olugbenga Fadipe is a synthetic organic chemist (chief research officer (CSO) ) with over twenty five years work expertise in policy making in all area of science & technology development particularly in venture capital management, drug repositioning and academic sectors. He is currently a research fellow in chemistry (organic/natural product chemistry) in the department of chemistry, university of Zululand, South Africa. His research interest is in the bioprosecting for drug lead candidates for infectious diseases from medicinal plants. He is a member of several learned society in chemistry.

Curtisia dentata is traditionally used in African traditional medicine to treat and manage sexually transmitted infections, diarrhoea, stomach complaints and as a purgative. C. dentata leaves were collected from Buffelskloof Private Nature Reserve in Mpumalanga province, South Africa. The ethanol, chloroform, ethyl acetate and acetone extracts were evaluated for antimicrobial activity using micro dilution assay against ATCC strains of Escherichia coli, Pseudomonas aeruginosa, Mycobacterium smegmatis, Mycoplasma hominis, Candida albicans and some clinical isolates of Moraxella catarrhalis, Proteus mirabilis and Staphylococcus aureus isolated from HIV patient. Acetone extract exhibited lowest MIC of 0.01 mg/ml against Candida albicans compared to other extracts. Besides lupeol, betulinic acid and ursolic acid, β-sitosterol was isolated for the first time from C. dentata leaves and exhibited moderate antimicrobial activity with MIC values ranging from 0.20 to 6.25 mg/ml. Furthermore, the ethanol extract and the four isolated compounds revealed MIC index of less than 4 suggesting that their effect is bactericidal. The ethanol extract revealed the best total activity of 2400 ml/g against Mycoplasma hominis compared to other extracts. Lupeol exhibited low selectivity index indicating that the compound had the similar cytotoxicity and antimicrobial activity. The cytotoxicity of the isolated compounds was further investigated against the human embryonic kidney (HEK293) and human hepatocellular carcinoma (HepG2) cell lines using the MTT assay. Ursolic acid exhibited the lowest LD50 of 122.4 µg/ml against HEK293 cell line while lupeol exhibited LD50 of 278.8 and 289.4µg/ml against HEK293 and HepG2 respectively. The ethyl acetate and acetone extracts were further investigated for antioxidant activity against 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The acetone extract exhibited 53.6% inhibition against DPPH at a concentration of 1 mg/100 ml. The findings of the current work validate the use of the plant species in the treatment of various human infections. The biological activity of the extracts, particularly the ethanol extract, may possibly be attributed to the isolated compounds. There is a need to explore the biological activity of the derivatives of the compounds isolated against other human pathogenic strains.

B Vijayalakshmi Ayyar received her M.Sc. in Biotechnology from the Barkatullah University, India in 2006 with her thesis on molecular diagnosis of PCV and PPV. She completed Ph.D. from Dublin City University, Ireland in 2011. Her Ph.D. research focused on developing high affinity antibodies against cardiac biomarkers and further validating them in various assay formats. Subsequently, Dr. Ayyar joined as Postdoc under Dr. Richard O’Kennedy and worked on generation of immunoreagents for cancer diagnostics. In 2012, she joined Dr. M.Z. Atassi’s Lab at Baylor College of Medicine, USA where she is working on identifying reliable vaccine candidates for botulinum neurotoxin A.

Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT poisoning occurs in a highly programmed fashion. After BoNT binds to cell surface, it is internalized, its light chain (LC) translocated into the cell. LC is a Zn-endopeptidase which causes enzymatic cleavage of SNARE proteins, thus inhibiting synaptic exocytosis. Over past two decades we investigated the immune recognition of BoNT/A and defined the epitopes on its heavy (H) and LC chains. In the current work, we explored significance of BoNT/A heavy chain N-terminal (HN) domain as a vaccine candidate. For evaluating protection, we used a mouse protection assay (MPA), which is considered the gold standard for BoNT toxicity assays. Mice were immunized with recombinant BoNT/A HN519-845. Antibodies (Abs) against HN519-845 protected mice against lethal dose of BoNT/A. Immuno-dominant regions of HN519-845 were identified and investigated individually for Ab response. Synthetic peptides covering HN519-845 were also studied for protective activity using MPA. Results were confirmed by patch-clamp analysis. Anti-HNAbs were studied for ability to block toxin-induced channel formation. The data strongly indicated that HN519-593 is an important region in generating protective Abs and should be valuable in a vaccine design.

Adebolu T T completed her PhD in Microbiology at Obafemi Awolowo University, Ile- Ife, Nigeria. Her research area is medical microbiology (Infections and Immunity). She has more than 50 papers in reputed journals. She served as the Editor in Chief for the School of Sciences Journal of Research in Science and Management and also as the Head, Department of Microbiology for six years at her University.

The effect of fermented cheese whey on the cells of the immune system and the packed cell volume (PCV) of apparently healthy albino rats (AHARs) was investigated in this study. Twenty four AHARs were grouped into six groups of four rats (A, B, C, D, E, F) and each rat in different groups was orogastrically administered different volumes of whey fermented at 30+2°C for 72 h as follows: group A; 0.5 ml, group B; 1.0 ml, group C; 1.5 ml, group D; 2.0 ml, group E; 2.5 ml per rats respectively while the rats in group F were not given whey. The rats in this group (F) served as control. All the rats were fed with basal diet alongside the administered whey except the control group that was given only the basal diet. Heamatological assays were carried out by collecting the rats’ blood through cervical dislocation into EDTA bottles and then analyzed using standard methods. The blood of the rats was evaluated for total White Blood Cells (WBC), differential WBC counts and Packed Cell Volume (PCV). The study revealed that the administration of the fermented whey at 2.0 ml and 2.5 ml per rat respectively caused a significant increase (p<0.05) in PCV values, lymphocyte and monocyte counts of the groups fed with whey as compared with control. It is therefore conceivable that the consumption of fermented whey by apparently healthy individuals might boost their immune system and increase their PCV.

TNF- plays a very important role in the progression of various inflammatory diseases including rheumatoid arthritis. This study aims towards the designing, synthesis and evaluating the effects of the peptide inhibitors of TNF-. Trimerized form of TNF-α is the active one, which can bind to receptor resulting in the progression of inflammatory cascade. The phenomenon of trimer formation was targeted for designing the inhibitors. The peptides were designed in silico using various bioinformatic tools and synthesized by solid phase peptide synthesis (SPPS) process. The anti-TNF activity of four different peptides (peptide 1, 2, 3 and 4) was checked in-vitro. Effect of peptide-2 was found to show the most significant anti-TNFα activity. TNF-α induced cell death of Wehi-164 cells was evaluated through MTT assay. The peptide-induced inhibition of TNF-a resulted in reduced cell death and increased viability. The TNF-α induced nuclear translocation of NF-kB was visualized using immune cytochemical analysis using A549 cells. The Western blotting of the nuclear lysate showed under expression of NF-κB in the TNF-stimulated cells with peptide inhibition as compared to the cells untreated with peptides. This result was fairly corroborated with the Electrophoretic Mobility Shift Assay (EMSA) test of the nuclear lysates of the experimental cells. Peptide-2 showed a dose dependent inhibition on the TNF-α activity with maximum effect at 200μM of the peptide concentration (p<0.05). No significant cytotoxicity was observed when stimulated with the peptides with the concentrations ranging between 50 and 400 µM. To conclude, peptide-2 showed the highest anti- TNF activity among all the peptides tested and this peptide-2 appears to have potential of being a peptide drug, for which in-vivo studies and further trials are required.

Satyendra Kumar Singh is pursuing his PhD from Sanjay Gandhi Postgraduate Institute of Medical Sciences, India. He has published seven publications in the peer- reviewed international journals. His paper presented in 4th National Conference of The Indian Academy of Tropical Parasitology- 2010, was awarded the prestigious “The Best Oral Paper Prize- 2010”. The title of his PhD work is “Immunopathogenesis of neurocysticercosis in naturally infected swine”.

Neurocysticercosis is caused by Taenia solium larvae. It is the most common helminthic infection in human. It causes variable clinical manifestations. Albendazole (ABZ) is the drug of choice for Taenia solium infection. So, we studied the local immune response operation at viable and degenerating cysts with and without albandazole treatment. Cysticercotic swine (n=30) were subjected to magnetic resonance imaging (MRI). Swine were divided in to three groups; group 1 received no treatment, group 2 treated with ABZ and group treated with ABZ plus steroid (ABZS). Group 2 and 3 underwent follow-up MRIs at 6 and 12 weeks and then sacrificed. Tissues surrounding the cysticerci were collected and studied for the expression of adhesion molecules, chemokines and matrix metalloproteinases by qRT-PCR and ELISA. Gel zymography was done to check the activities of MMPs. Genetic characterization of cysticerci was done using cox1 gene. Genetic characterization showed the Asian genotype of T. solium with minimal genetic variation. We also reported for the first time about the existence of T. asiatica in India. In group 1, the expressions of ICAM-1, E- selectin, MIP-1, RANTES and EOTAXIN-1were associated with degenerating cysts whereas MMP-2, -9, VCAM-1 and MCP-1 were associated with viable cysts. In group 2 and 3, ABZ therapy was found to be more effective in parasite killing than ABZS. In group 2, all cysts responded ABZ therapy and induced the expression all above immune markers except VCAM-1 and MIP-1 around the dead cysticerci. In group 3, despite a heavy parasite burden in the brain, all the pigs treated with ABZS survived. MMP-9, ICAM-1, E-selectin and RANTES expressions were associated with dying/ dead cyst when compared to viable cysts.

Spinal cord injury (SCI) elicits a robust intra-spinal inflammatory response with potentially devastating consequences. Immune cells present in the injury site may sequester cell debris and carry spinal antigens (SAgs) to secondary lymphoid organs. There, SAgs may be processed and presented by antigen presenting cells to lymphocytes, triggering lymphocyte activation. In clinical and experimental SCI, only a few autoantigen targets have been documented. ACAID (anterior chamber associated immune deviation) is an antigen-specific form of peripheral immune tolerance (IT) that is induced against exogenous antigens placed in the anterior chamber (AC) of the eye. It is characterized by the inhibition of delayed hypersensitivity reactions to the AC-injected antigens. This IT is elicited by AC-induced CD4+ CD25+ antigen-specific regulatory T cells. Since neural degeneration after SCI includes a strong inflammatory component triggered by reactivity against multiple antigens derived from the neural tissue, this project proposes to induce IT against SAgs as a neuro-protective strategy in SCI. Results to date indicate that it is possible to induce IT to a cocktail of SAgs obtained from healthy rat spinal cord by ACAID. Neuroprotection of the spinal cord will be evaluated by histological and immuno-fluorescence techniques and further functional evaluations will be done with sensorimotor tests as indicators of spinal cord tissue preservation.

The use of therapeutic immune cell subsets (aka cell therapy) is becoming an increasingly cost-effective and attractive strategy for the treatment of cancer. Specialized cell subsets including dendritic cells (DC), cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, cytokine-induced killer (CIK) cells, tumor infiltrating lymphocytes (TIL), and genetically engineered chimeric antigen receptor (CAR) T-cells have all shown efficacy, at times significant efficacy, in a variety of different clinical studies. Nonetheless, the field of cell therapy is hampered by the inability to consistently generate and expand sufficient numbers of high quality cells from a majority of patients. To address this critical issue, the investigators have developed a sophisticated bioreactor technology (Published patent information: PCT/US2012/000182) that maximizes metabolic support with minimized shear-stress forces, automatically monitors functional correlates to allow cell harvest at peak functional capacity, and permits cell sorting in situ to minimize cell loss and microbial contamination. This revolutionary technology has generated increased numbers of high quality hematopoietic stem cells for use in hematopoietic stem cell transplantation and has also demonstrated the capacity to generate enhanced numbers of functionally superior immune cell subsets. In current study, splenocytes from cancer cell-primed BALB/c mice were isolated and seeded in a ZYX Bioreactor (ZYX btr) cell culture chamber and 6-well plates at 106 /ml and cultured for 6 days with an RPMI containing mouse serum, IL-2, IL-7, and irradiated cancer cells (Renca cells, seeding density determined by surface area, 80% confluent). On day 6, suspension cells in the ZYX btr were processed for CD8+ cell isolation using a positive selection program and then cultured with the same medium for 3 more days. CD8+ cells in a static culture were isolated using a Miltenyi device and then placed back in plate to culture with the same medium for 3 more days. Following the expansion, 2x, 4x, or 8x105/well effector cells were then added into 25,000/well target cells in triplicate and incubated for 4 hours. Supernatants were then harvested for LDH detection. CTL activity (% lysis) was calculated using the formula (Test cell mix-Effector control-spontaneous release)/(maximum release-spontaneous release). The data shows that when the effector cells were stimulated in the ZYX btr, the CTL expansion folds and overall lytic activity were significantly higher than those in the static culture (p<0.05). The ZYX btr significantly increased cancer-specific CTL production in the expansion, and the expanded CTLs in the ZYX bioreactor have higher killing activities in comparison with the static culture, which suggests that the ZYX bioreactor provides a better cell growth environment for antigen-specific CTL expansion than the static culture and will benefit cancer-immunotherapy.

Background
Immunity to malaria requires innate, adaptive immune responses and Plasmodium-specific memory cells. Previously, mice semi-immune to malaria was developed. Three cycles of infection and cure (3-cure) were required to protect mice against Plasmodium bergheiANKA infection.

Methods
C57BL/6J mice underwent three cycles of P. berghei infection and drug-cure to become semi-immune. The spleens of infected semi-immune mice were collected for flow cytometry analysis. CD11c(+) cells of semi-immune mice were isolated and transferred into naïve mice which were subsequently challenged and followed up by survival and parasitaemia.

Results
The percentages of splenic CD4(+) and CD11c(+) cells were increased in semi-immune miceon day 7 post-infection.The proportion and number of B220(+)CD11c(+) cells (plasmacytoid dendritic cells, DCs) was higher in semi-immune, 3-cure mice than in their naïve littermates on day 7 post-infection (2.6 vs 1.1% and 491,031 vs 149,699, respectively). In adoptive transfer experiment, three months after the third cured P. berghei infection, splenic CD11c(+) DCs of non-infected, semi-immune, 3-cure mice slowed Plasmodium proliferation and decreased the death rate due to neurological pathology in recipient mice. In addition, anti-P. berghei IgG1 level was higher in mice transferred with CD11c(+) cells of semi-immune, 3-cure mice than mice transferred with CD11c(+) cells of naïve counterparts.
Conclusion

CD11c(+) cells of semi-immune mice protect against experimental cerebral malaria three months after the third cured malaria, potentially through protective plasmacytoid DCs and enhanced production of malaria-specific antibody.

Abdelhalim has completed his MBBS on Jan,2012 at the age of 25 years from Omdurman Islamic University and diploma of molecular biology from Sudanese center of biotechnology on March,2013 . He is a junior registrar in training programme leading part one examination in Clinical Immunology at Sudan Medical Specialization Board . He has published one paper related to detection of genes defect through using software tools.

A 35-year-old female was admitted to our hospital(Wad Medani,Gazera state ,Sudan) on April 10, 2015 because of 3years history of generalize weakness, and multiple joint pain and swelling mainly wrist and knee joint ,recently all joints were involved. After 2 months later patient developed high grade, intermittent fever mainly at night . Physical examination revealed swollen hands with bilateral rheumatoid deformities(swelloing,hottnes,pain,restriction of movement) , nose scleroderma, small mouse and nasal septum perforated which leaded to bloody sputum .Patient also have sclerodactyly and heliotrope rash but there is no Raynaud's phenomenon. The body temperature was fluctuated but almost times was febrile. Laboratory data showed thrombocytopenia(62x103) and anemia(RBCs 2.61x1012,HB 6.9 g/dl. Serological examination revealed ESR 130,RF positive more than 8 Iu/ml , and anti-RNP antibody was positive,Anti-ds DNA +ve,. Neither anti-DNA antibody nor anti-Sm antibody were detected .Also Serum C3 and C4 not detected . Treatment with prednisolone 10mg (2 tabs at morning and 2 tabs at evening) daily was started. Beside , Refina ( as prophylaxis for TB,so she has a history of chronic cough ) 300 mg ,2 tabs morning.Azathioprine 50 mg once daily. Next day the treatment with 1000-mg intravenous daily pulse of methylprednisolone for 3 days was started, After this pulse therapy, ten days after the pulse therapy joint examination revealed better and painful was subsided and laboratory data showed improvement as RBCs changed to 4x1012 , platelet changed to 112x103 and, HB to 10 g/dl . Prednisolone was slowly tapered to 20 mg daily.

Introduction: Human γδ T cells display potent cytotoxicity against various tumor cells pretreated with zoledronic acid (Zol). Zol has shown benefits when added to adjuvant endocrine therapy for patients with early-stage breast cancer or to standard chemotherapy for patients with multiple myeloma. Although γδ T cells may contribute to this additive effect, the responsiveness of γδ T cells from early-stage breast cancer patients has not been fully investigated. Objective: In this study, we determined the number, frequency, and responsiveness of Vγ2Vδ2 T cells from early- and late-stage breast cancer patients and examined the effect of IL-18 on their ex vivo expansion. Methods: Breast cancer patients (n=80) were enrolled after institutional review board approval and with written informed consent. Peripheral blood mononuclear cells (PBMC) were purified and stimulated with Zol/IL-2 or Zol/IL-2/IL-18 for 2 to 10 days. The expanded cells were assessed on flow cytometry and the production of IFN-γ and TNF-α measured through ELISA. Results: The responsiveness of Vγ2Vδ2 T cells from patients with low frequencies of Vγ2Vδ2 T cells was significantly diminished. IL-18, however, enhanced ex vivo proliferative responses of Vγ2Vδ2T cells and helper NK cells (CD3-CD56brightCD11c+CD14-CD16+NKGD2+NKp44low) from patients with either low or high frequencies of Vγ2Vδ2 T cells. Cell-to-cell contact between γδ T and helper NK cells appeared to promote expansion of γδ T cells. Exogenous IL-18 markedly enhanced IFN-γ and TNF-α production from PBMC stimulated by Zol/IL-2, whereas the addition of an anti-IL-18Rα mAb reduced cytokine production. Conclusion: These results demonstrate that Zol elicits immunological responses by γδ T cells from early-stage breast cancer patients and IL-18 enhances proliferative responses and effector functions of γδ T cells in the context of helper NK cells.

Daniel G Rey Caro is a Staff member of the ENT Service of Chutro Clinic in Cordoba, Argentina, Member of Argentine Society of Otolaryngology and Children Phonoaudiology, Member of Argentine Society of Voice, Member of Argentine Federation of Societies of Otolaryngology and a Member of Cordoba Society of Otolaryngology.

Introduction: Chromoendoscopy is an endoscopic technique which uses a contrast stain to paint the aerodigestive tract mucosal lining followed by an optical assessment to highlighting any epithelial abnormalities. Detailed and high-definition magnified views achieved with the aid of rigid endoscopes can often allow for identification of the tissue type or pathology based upon the pattern uncovered. According to the literature we reviewed, we may have been the first ones to use indigo carmine in the field of otolaryngology. Tiny lesions that usually go overlooked with conventional microlaryngoscopy become visible upon the instillation of indigo carmine and further decreasing the chances of an early lesion postoperative recurrence. Chromoendoscopy in recurrent respiratory papillomatosis (RRP) helps identify unsuspected intraoperative lesions by clearly enhancing the view of their boundaries and surface type. It is also suitable to assess the presence of residual lesions, if any, after their surgical removal. Objectives: To demonstrate the usefulness of chromoendoscopy in RRP in laryngotracheal surgery. Material & Methods: We used indigo carmine associated with endoscopic laryngeal surgery. Before staining, the mucosa may need to be treated with a mucolytic agent to get rid of excess mucus to boost staining. Rigid suspension laryngoscopes of different proximal and distal diameters were used with chromoendoscopy. Patients underwent chromoendoscopy associated with endoscopic laryngeal surgery under general anesthesia in the O.R. Results: In this second phase of our research work, this diagnostic technique was applied to eighteen patients with recurrent laryngeal papillomatosis and two patients with suspected carcinoma of the larynx. We were able to optimize the intraoperative diagnosis and reduce the likelihood of the relapse risk in all patients. Conclusion: Chromoendoscopy associated with endoscopic laryngeal surgery is an excellent low-cost intraoperative diagnostic method for the treatment of invasive diseases of the larynx such as laryngeal papillomatosis.