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Problem: Studies have shown a relationship between endometriosis and ovarian cancer. Our aims were to evaluate and compare the dosages of cytokines IL-2, IL-5, IL-6, IL-8, IL-10, and TNF-α in serum, intracystic fluid, and peritoneal fluid of patients with ovarian endometrioma, malignant and benign ovarian neoplasms, and non-neoplastic ovarian tumors; to verify if there is a correlation between the values of these cytokines between ovarian endometrioma and ovarian malignancy; and to determine the best cut-off point for serum cytokines that can be used to differentiate patients with ovarian malignancy and endometrioma., Method of Study: The concentrations of cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), analyzed by Kruskal-Wallis test with the Dunn post-test. Receiver operating feature (ROC) curve was used to obtain the area under the curve (AUC) and to determine the best cut-off values that could be used in the diagnosis of ovarian malignancy. Correlations of cytokine concentrations were performed by the Spearman test., Results: IL-6, IL-8, and IL-10 concentrations were higher in patients with malignant neoplasia. When evaluating the area under the curve (AUC) of serum cytokine levels comparing patients with malignant neoplasia and endometriomas, there was statistical significance for IL-6, IL-8, and IL-10., Conclusion: Our results showed utility in serum concentrations of IL-6, IL-10, and IL-8 as parameters that differentiate endometriomas from ovarian malignancies., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: The aim was to investigate the systemic levels of cytokines and the expression of the chemokine receptor CXCR2 in circulating neutrophils in patients with non-neoplastic ovarian lesions, benign neoplasia or malignant neoplasia., Materials and Methods: Controls and patients with ovarian tumours were pre-operatively compared for the production of cytokines (IL-2, IL-5, IL-6, IL-8, IL-10 and TNF-α) by ELISA, and for the expression of the chemokine receptor, CXCR2, in neutrophils, by flow cytometry. Randomly selected patients within the malignant group were re-evaluated for the inflammatory parameters at 30 days after surgery., Results: The serum concentrations of IL-6, IL-8 and IL-10 were significantly higher in the benign and malignant neoplasia than in the control group, and their levels were significantly higher in ovarian cancer patients than in patients with non-neoplastic tumours or benign neoplasia. Treatment reduced IL-8 serum levels but did not affect CXCR2 expression in neutrophils. Cut-off values for IL-6, IL-8, and IL-10 comparing malignant vs. benign neoplasia were 11.3, 71.7, 14.8, and comparing malignant neoplasm vs. non-neoplastic lesions were 7.2, 43.5, 12.3, respectively., Conclusions: Serum IL-6, IL-8, and IL-10 levels, and expression of CXCR2 in circulating neutrophils seem promising for distinguishing ovarian cancer patients from patients with benign tumours.

The objectives of the study were to analyze the dosage of a cytokine panel (IL2, IL5, IL6, IL8, IL10, and TNF-α) in the peritoneal fluid and relate the dosage of these cytokines to prognostic para- meters and survival in ovarian cancer. Peritoneal fluid was collected intraopera- tively from 29 patients with primary malignant ovarian neoplasia. Cytokine panel dosing was performed with ELISA. Comparisons of cytokines with prognostic factors were performed using the Wilcoxon-Mann-Whitney test. ROC curves were used to determine the cutoff value of NLR, PLR, and IL6. Univariate and multivariate analysis of disease-free survival (DFS) or overall survival (OS) were performed (Kaplan-Meier and Cox regression). The differences were considered significant when the value of p < .05. Higher levels of IL-6 were related to a neutrophil-lymphocyte ratio (NLR) >3.18 (p = .04), a platelet-lymphocyte ratio (PLR) >219.23 (p = .0051), CA-125 levels >35 U/mL (p = .0019), stage IIIC (p = .0203), and DFS ≤ 24 months (p = .0267). For IL-8, higher levels were related to PLR > 219.23 (p = .0426), and CA-125 >35 U/mL (p = .0292). In the univariate analysis, IL-6 levels ≥87.23 in peritoneal fluid had a relationship with shorter DFS at significance threshold (p = .05), and with a shorter OS (p = .039). Multivariate survival analysis proved that IL-6 level in the peritoneal fluid was an independent predictor of OS. Therefore, IL-6 and IL-8 in peritoneal lavage were related to poor prognostic factors. These cytokines may represent new biomarkers for ovarian cancer therapies.

Objective: The objectives of the present study were to demonstrate the influence of neutrophils, platelets, and neutrophil-lymphocyte ratio (NLR) in the ovarian cancer prognosis and to compare these parameters with benign ovarian neoplasms., Materials and Methods: Records of patients underwent surgery for ovarian cysts were evaluated. There were 72 malignant neoplasms and 213 benign neoplasms. Age, parity, histologic type, tumor stage, type of surgery performed, chemotherapy, disease-free survival, serum levels of tumor markers, neutrophils, lymphocytes, platelets, and NRL were recorded. The Mann-Whitney, the Chi-square test and multiple linear regression were used. A P-value < 0.05 was established as the significance level., Results: Higher values of platelets, neutrophils, and NLR were found in malignant tumors (P = 0.0132, P = 0.0208, and P < 0.0001, respectively), while lymphocytes values were higher in benign group (P < 0.0001). Preoperative platelet count 300,000/mm3 was related to less aggressive histological types (P = 0.0148). NLR <3 was related to the initial stages (P = 0.0053), and patients with disease-free survival >24 months had most often neutropenia during chemotherapy (P = 0.0482). After multivariate analysis, platelets, NLR, and serum levels of CA15.3 were considered independent variables related to tumor staging (P = 0.028, P = 0.028, and P = 0.035, respectively)., Conclusion: NLR and serum levels of platelets may represent potential prognostic factors in ovarian cancer, and they may also serve as therapeutic targets in the future treatment strategies., Competing Interests: None

The aims of the study were to compare the levels of tumor necrosis factor alpha (TNF-α) and its soluble type I (sTNF-R1) and type II (sTNF-R2) receptors detected in intracystic liquid and serum from benign and malignant ovarian neoplasms and to relate them to prognostic factors in epithelial ovarian cancer. The patients were divided into benign ovarian neoplasms (n = 46) and malignant ovarian neoplasms (n = 17). The serum and intracystic samples were collected before and during surgery for ovarian cyst, respectively. The levels of TNF-α, sTNF-R1, and sTNF-R2 were measured using ELISA. Results were compared with the Mann-Whitney test. Concentration of sTNF-R2 in the intracystic samples collected from the malignant neoplasia was significantly higher than that of the benign neoplasias (p = 0.02). Higher intracystic levels of sTNF-R2 exhibited a significant association with tumor differentiation grades 2 and 3 (p = 0.0087). There was no statistical significance in relation to serum levels. Tumor microenvironment levels of sTNF-R2 may represent a factor of poor prognosis in epithelial ovarian cancer.

Background/aims: Growth differentiation factor 15 (GDF-15) is induced by pro-inflammatory cytokines. Higher levels of GDF-15 have been associated with malignancy. The aim of the study was to evaluate both tissue and serum levels of GDF-15 in ovarian neoplasms., Methods: A cohort study evaluated 31 patients with benign ovarian tumors and 34 patients with ovarian cancer were evaluated in 2 years. The inclusion criterion was histopathological diagnosis of ovarian epithelial neoplasia. Exclusion criteria were secondary malignant ovarian neoplasia and preoperative treatment. Serum and tissue levels of GDF-15 were assessed by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Chi-square test and unpaired t test were performed., Results: Serum levels were higher in the patients with malignant neoplasms than in the patients with benign tumors, yet the difference was not statistically significant. GDF-15 immunostaining was significantly more frequent in the stroma of the malignant tumors than in the stroma of the benign tumors (p = 0.0034)., Conclusion: GDF-15 staining is elevated in the stroma of ovarian cancer, demonstrating that it may be a potential diagnostic and therapeutic target., (© 2017 S. Karger AG, Basel.)

The development of new biomarkers for the diagnosis and prognosis of ovarian cancer may provide an opportunity for new therapies. In this study, we aimed to compare cytokines (interleukin [IL]-2, IL-5, IL-6, IL-8, IL-10 and tumour necrosis factor [TNF]-α) and nitric oxide (NO) metabolite levels in non-neoplastic tumours, benign primary ovarian tumours and malignant primary ovarian neoplasms. The secondary aim was to relate cytokine and intracystic NO metabolite levels to clinical, laboratory and pathologic characteristics for patients with primary ovarian malignancies. We evaluated 110 patients with adnexal masses. Cytokine concentrations were quantified by enzyme-linked immunosorbent assay and nitrate concentrations by enzymatic reduction of nitrite by nitrate reductase. Patients with malignant neoplasms had higher IL-6, IL-8 and NO levels compared to patients with benign neoplasms. Histologic grade 1 tumours were associated with elevated IL-2 levels, whereas anaemia was associated with elevated IL-6 levels. On average, those patients with elevated IL-8 levels also had a neutrophil/lymphocyte ratio (NLR) greater than 2.6 and less than 36 months of disease-free survival (DFS). Patients with normal CA 19-9 levels had elevated IL-10 levels. TNF-α was elevated in patients with two carcinogenesis and those with a platelet/lymphocyte ratio (PLR) less than 300. NO levels were higher in patients with an NLR less than 2.6 and CA 19-9 greater than 35 U/ml. Elevated intracystic cytokine levels, especially IL-6 and IL-8, are associated with worse prognosis in ovarian cancer., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)

The aim of the study was to correlate serum levels of IL-2, IL-5, IL-6, IL-8, IL-10, and TNF-α with clinical, laboratory, and pathological prognostic factors in patients with primary ovarian malignancy. Patients treated at the Pelvic Mass Ambulatory of the Discipline of Gynecology and Obstetrics/Oncology Research Institute (IPON) of the UFTM with confirmed diagnosis of malignant ovarian neoplasia (n = 26) were evaluated. Serum collection was performed preoperatively for the determination of tumor markers. The cytokines IL-2, IL-5, IL-6, IL-8, IL-10, and TNF-α were assayed by enzyme-linked immunosorbent assay (ELISA). The prognostic factors were compared using the Mann-Whitney test, with significance level lower than 0.05. When evaluating IL6, it was observed that higher serum levels were associated with overall survival less than 60 months (p = 0.0382). In the evaluation of IL8, higher serum levels were associated with neutrophil-to-lymphocyte ratio (NLR) ≥ 4 and platelet-to-lymphocyte ratio (PLR) ≥ 200 (p = 0.0198 and p = 0.0072, respectively), altered values of serum CA125 (p = 0.0457), and stage IIIC (p = 0.0486). Therefore, increased levels of IL-6 and IL-8 are associated with factors of worse prognosis in ovarian cancer. Additional studies with a larger sample of patients are needed to confirm the role of cytokines as prognostic factors, in the definition of treatment, and in the development of future target therapies.

Cetuximab is a chimeric monoclonal antibody that acts as a competitive antagonist, by binding to EGFR. This cell signalling pathways regulates tumor progression. The oral squamous cell carcinoma undergoes to regional spreading and distant metastasis. This study aimed to evaluate the effect of treatment with Cetuximab on cell migration and invasion in OSCC cells, by using the SCC-4 cell line. Cell migration and cell invasion assay were performed and actin cytoskeleton of control and treated with Cetuximab cells were evaluated. Differences were considered significant when p&lt;0.05.Cetuximab inhibited the migration of SCC-4 cells at three concentrations: 1 µg/mL, 50 µg/mL and 100 µg/mL (p&lt;0.0001) in a dose-dependent manner. The number of SCC-4 treated cells with 1 μg/mL that migrated through the membrane was statistically different from 50 μg/mL (p&lt;0.001) and 100 μg/mL (p&lt;0.0001), and between 50 μg/mL and 100 μg/mL (p&lt;0.01). Cetuximab 50 μg/mL inhibited cell invasion through the MatrigelTM compared with SCC-4 control cells (p&lt;0.01). Cetuximab 50 μg/mL affected the organization of the actin cytoskeleton. Cetuximab has an inhibitory effect on actin cytoskeleton organization, cell migration and invasion, suggesting that Cetuximab treatment can be important to avoid oral squamous cell carcinoma metastasis.

Purpose of Investigation: The aims of study were to evaluate potential prognostic laboratory factors in ovarian cancer (blood count parameters and tumor markers) and to relate these parameters with prognostic factors., Materials and Methods: The authors evaluated patients that underwent surgical treatment and with confirmed histopathologic diagnosis of ovarian cancer. Age, FIGO stage, type of surgery, serum levels of tumor markers, parameters of blood count, disease-free and overall survival were recorded. Mann-Whitney test was performed. The significance level was less than 0.05., Results: Higher levels of CA 125, CA 15.3, and platelets were found in the group with Stage hII/I, since hemoglobin levels were higher in stage I/II (p < 0.05). CEA levels were higher in the group of non-serous neoplasms (p < 0.05). Higher levels of CA125, CAIl5.3 and platelets were seen in the group histological grade 2/3 (p < 0.05)., Conclusion: CA 125, CA 15.3, hemoglobin, and platelets can be related prognostic factors in ovarian cancer.

Objective: To characterize the inflammatory profiles of patients with systemic lupus erythematosus receiving standard treatment compared to healthy controls., Patients and Methods: Peripheral venous blood was collected from systemic lupus erythematosus patients (n=14) and controls (n=18) at enrollment. Blood samples were used for quantification, by flow cytometry, of CD11b (integrin) and Chemokine receptor CXCR2 expression surface antigen in neutrophils and lymphocytes, while cytokines were assayed in serum samples. Purified neutrophils were assayed by their ability to phagocytize human plasma-opsonized zymosan., Results: Patients had a median (interquartile range) disease activity index of 1.0 (0-2.0) characteristic of patients in remission. Interleukin-6 and interleukin-10 serum concentrations were significantly higher in the patient group compared to controls and the phagocytic index of circulating neutrophils was significantly reduced in patients compared to controls. The levels of interleukin-2, interleukin-5, interleukin-8 and tumor necrosis factor alpha did not significantly differ between patients and controls. Flow cytometric analysis revealed that the integrin expression levels were reduced in lymphocytes (but not in neutrophils) obtained from systemic lupus erythematosus patients, while surface expression of the chemokine receptor 2 was similar in both neutrophils and lymphocytes., Conclusion: Systemic lupus erythematosus patients receiving standard treatment presented with elevated systemic levels of interleukin-6 and interleukin-10, reduced neutrophil phagocytic capacity, and reduced lymphocyte expression of integrin even when symptoms were in remission. These alterations to innate immune components may put these individuals at a greater risk for acquiring infections., (Copyright © 2016 Elsevier Editora Ltda. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is the most malignant lesion occurring in the head and neck. The Rho-kinases (ROCKs), effectors of Rho proteins, are involved in actin cytoskeletal organization, cell migration, and maintenance cortex. The HA-1077 inhibits the ROCKs. This study aimed to evaluate the effect of treatment with HA-1077 on cell motility in SCC-4 cells, a cell line originating from human OSCC. F-actin of SCC-4 cells treated or not with HA-1077 (1, 50 and 100 μmol/l), and also HA-1077 50 μmol/l and/or inhibitors Y-27632 30 μmol/l was stained with rhodamine-conjugated phalloidin and analyzed by confocal microscopy. Approximately 1×10 cells/well, control and treated with HA-1077 (25, 50, and 100 μmol/l) were added to the migration plate assay. In addition, 1×10 cells/well, control and treated with HA-1077 50 μmol/l, were tested by invasion assays (plate coated with Matrigel). The inhibition of ROCKs with HA-1077 and/or Y-27632 leads to morphological changes, affecting the organization of the actin. The inhibitory effect of HA-1077 (P<0.0001) was dose dependent as the number of cells migrated at 100 μmol/l was statistically different: 25 μmol/l (P<0.0001) and 50 μmol/l (P<0.01). The number of cells treated with HA-1077 50 μmol/l decreased compared with control cells that invaded through Matrigel (P<0.0001). This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC.

Purpose: To investigate the mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy., Methods: Peripheral venous blood was collected at the time of diagnosis and immediately prior to the 4th cycle of an anthracycline-based chemotherapy regimen for patients diagnosed with different stages of breast cancer (n = 30), for experimental assays. Blood samples were also collected from a healthy control group (n = 17)., Results: IL-8 serum concentrations were higher in the patient group than in the control group (p = 0.02), and chemotherapy did not further affect this increase. Levels of TNF-α, IL-6, and IL-10 did not differ between controls and patients, or in relation to chemotherapy. Serum levels of nitric oxide (NO) metabolites were elevated following chemotherapy compared to levels detected prior to treatment (p = 0.01). When the supernatants of lipopolysaccharide-stimulated mononuclear cells and neutrophils obtained from the patients were assayed for levels of nitrite, these levels were significantly higher and unchanged, respectively, compared with controls. Expression levels of the chemokine receptors, CXCR1 and CXCR2, were significantly reduced in patients compared to controls, and chemotherapy did not further affect these differences. Furthermore, filamentous actin content for IL-8-activated neutrophils was reduced with chemotherapy (median 8.85; range 3.38-13.43) compared to the content detected prior to treatment (median 9.23; range 2.86-22.16) (p = 0.001)., Conclusion: Elevated systemic levels of IL-8 and NO, desensitization to CXCR activation, and reduction in actin polymerization may affect neutrophil motility in patients before and after chemotherapy.

The aim of this paper was to evaluate the parameters of blood count and tumor markers in borderline ovarian tumors. We evaluated 21 patients who had confirmed histopathologic diagnosis of borderline ovarian tumor. We recorded age, parity, tumor type, stage of cancer, serum levels of tumor markers (CA-125, CA-15.3, CA-19.9, CEA, AFP), and the parameters of blood count, fasting glucose, disease-free survival and overall. The patients were divided into two groups, stage IA (n = 13) and stage IB-IIIC (n = 8). The unpaired t-test and Fisher's exact test were used, with P values of less than 0.05 being considered to indicate statistical significance. Levels of red blood cells, hematocrit, and hemoglobin were significantly higher in stage IA when compared with stage IB-IIIC (P < 0.05). The levels of tumor marker CEA had a tendency to be higher in the group stage IB-IIIC (0.08). Abnormal levels of CEA and CA-19.9 were found more frequently in stages IB-IIIC. Therefore, parameters of blood count, CEA, and CA-19.9 should be targeted for further research in identifying prognostic factors in borderline tumors.

Aim: To verify if patients with cervical neoplasia produce mediators that reduce leukocyte function., Methods: Control neutrophils incubated with normal serum or serum from pre-invasive or invasive neoplasia patients were assayed for chemotaxis. Mediators were assayed in serum and in leukocyte supernatants. Experiments were also performed in random patients after surgery., Results: Neutrophils incubated with patient sera, but not normal sera, failed to migrate towards the chemoattractants. In invasive neoplasia compared to controls, IL-6 and IL-8, and IL-10 and TNF-α were elevated in serum and in neutrophil supernatants, respectively. Nitrite levels were elevated in mononuclear cell supernatants from patients than controls. After surgery, serum cytokine levels were reduced, mainly in pre-invasive patients. Neutrophils treated with serum from pre-invasive patients undergone surgery had restored migration., Conclusion: Patients with cervical neoplasia produce mediators, predominantly induced by tumor cells, able to impair the inflammatory response at very early stages of disease.

Objective: The aim of the study was to evaluate blood leukocyte counts in patients with uterine cervical neoplasia., Methods: Patients treated at a university hospital were reviewed retrospectively. Disease progression was monitored, beginning in 1990 to 2002, for at least 5 years. Blood count parameters included absolute leukocyte, neutrophil and lymphocyte counts, leukocytosis (white blood cells > 10³/μL), neutrophilia (neutrophils ≥ 70% of leukocytes), lymphopenia (lymphocytes ≤ 15% of leukocytes), and the neutrophil-lymphocyte ratio (NLR), categorized as less than 5 or 5 or greater., Results: A total of 315 patients were enrolled: 182 (57.8%) with preinvasive neoplasia (cervical intraepithelial neoplasia [CIN] group), 95 (30.1%) with stages I to II (early group), and 38 patients (12.1%) with stages III to IV neoplasia (advanced group). Neutrophil and lymphocyte counts were elevated and reduced, respectively, at advanced stages compared with the CIN group (P < 0.05). Leukocytosis, neutrophilia, lymphopenia, and an NLR of 5 or greater were more frequent at advanced stages compared with the CIN and early-stage groups (P < 0.05). Moreover, neutrophilia was also significantly more frequent at early stage compared with the CIN group. The advanced group with neutrophilia had increased frequency of recidivism and metastasis than patients in the CIN group with neutrophilia (P < 0.05)., Conclusions: Patients with advanced cervical cancer had significantly higher frequency of leukocyte alterations, although they may occur apart from the preinvasive stages. Overall, neutrophilia was the best indicator of cancer invasiveness.

The presence of chromosomal aberrations induced in circulating lymphocytes from breast cancer patients during chemotherapy was analyzed. Ten breast cancer patients undergoing neoadjuvant chemotherapy and ten healthy women (controls) were evaluated. Metaphases were obtained from cultures of peripheral lymphocytes stimulated with phytohemaglutinin and metaphase blockage was achieved with colchicine. One hundred metaphases were analyzed for chromosomal aberrations and 1,000 cells for the mitotic index. No significant differences were observed regarding the frequency of chromosomal aberrations, number of cells with chromosomal aberrations and mitotic index between the controls and patients before chemotherapy. However, after the first chemotherapy cycle, the numbers of chromosomal aberrations and cells with them was greater. After the third cycle, the mitotic index was lower, but the fifth cycle produced an increase in relation to the third and fourth cycles. The results suggest that chemotherapy raises the number of chromosomal aberrations and favors persistence of stable chromosomal abnormalities.

The aim was to evaluate the impact of anthracycline-based chemotherapy on neutrophil count and infections in breast cancer women. The medical records of patients were retrospectively and prospectively reviewed (8-year period). Patients were grouped according to anthracyclines at different doses: (1) Scheme 1 (n = 56, 224 courses): 50-60 mg/m(2); and (2) Scheme 2 (n = 25, 100 courses): 65-75 mg/m(2), associated to cyclophosphamide and 5-fluorouracil, at 21-day intervals between courses. Neutrophil count was performed on diagnosis and 48-72 h before each chemotherapy course. Patients were followed up for neutrophil count and infection episodes for three consecutive courses. Multivariate analysis was used to determine independent factors for infection. After the first course, neutrophil count was reduced than baseline (P < 0.001) and maintained during the subsequent courses, without differences between courses or groups. There were 49 infection episodes (63.2% urinary, 18.4% neutropenic fever and 18.4% diverses), mainly between course 1-2 (39%) and course 3-4 (38%) of chemotherapy. Patients evaluated as presenting or not with infection episodes did not differ in neutrophil count. The number of chemotherapy courses (P < 0.05), but not age, neutrophil count or chemotherapy regimen, was associated with infection. We concluded that progressive chemotherapy, but not neutrophil count, was an independent factor for infection.

Aims and Background: Our aim was to evaluate the effect of treatment on the in vitro migration of circulating mononuclear cells in cervical cancer patients at different stages., Methods: We prospectively investigated 24 patients with cervical neoplasia, without prior treatment, submitted to surgery or chemotherapy as therapeutic conduct. Controls were healthy volunteer women (n = 23). Mononuclear cells were isolated from peripheral venous blood before and after treatment, and their migration capacity was evaluated in a microchemotaxis chamber assay towards the chemotactic stimuli fMLP, MCP-1 and RANTES, compared to basal migration. Serum levels of nitric oxide metabolites were assayed by the Griess reaction., Results: Increased mononuclear cell migration in response to the chemotactic stimuli, compared to basal migration, was observed in controls and patients, without differences between them. After treatment (n = 14), mononuclear cell migration in response to MCP-1 and RANTES was increased compared to pre-treatment. Serum levels of nitric oxide metabolites were more elevated in patients (n = 19) than in controls (n = 17), but decreased after treatment (n = 15)., Conclusions: The results suggest that the production of soluble circulating factors by tumor cells could interfere with the functional activity of blood mononuclear cells.

Objective: To evaluate the local immune response in patients with bacterial vaginosis (BV) and cervical intraepithelial neoplasia (CIN), as assessed by cytokine and nitric oxide (NO) concentrations., Study Design: Patients attending for routine gynaecological examination were prospectively enrolled in groups: BV (n=25) diagnosed by clinical criteria, CIN graded I to III (n=35, 6 CIN I, 8 CIN II and 21 CIN III) by histological analysis, and controls (n=15) without clinical and cytological findings. Randomly selected patients within CIN group at grades II or III (n=15) were re-evaluated at 60 days after surgical treatment. Endocervical (EC) and vaginal secretion samples were collected by cytobrush and the levels of cytokines (ELISA) and NO metabolite (Griess reaction) were assayed., Results: NO was assessed in all subjects, and cytokines in all controls, 15 BV and 30 CIN patients. Interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and nitrite levels were higher in EC than in vaginal secretions in BV and CIN groups. In CIN group, IL-8, IL-10 and nitrite concentrations were greater in EC and/or vaginal secretions than in BV or controls. Surgical treatment reduced IL-8 levels in EC and vaginal secretions., Conclusion: A similar local immune profile was found in BV and CIN groups. The increased local production of IL-8, IL-10 and NO in CIN suggests a role for these mediators in the immune response against tumour or tumour development.

To determine parameters associated with the evolution of sepsis, a five-year retrospective study was conducted in a university hospital. One hundred and four consecutive sepsis patients were evaluated, of whom 55.8% were men. The mortality was 68.3% and was associated with older age (p<0.05). Chronic comorbidities and infection site were not associated with prognosis. Gram-positive bacteria were more frequently identified in survivors (p<0.05), while non-detection of the germ was associated with mortality (p<0.01). Appropriate use of antibiotics (germ sensitive to at least one drug administered) was associated with survival (p<0.0001) while inappropriate use (p<0.05) or empirical use (p<0.01) were more frequent in nonsurvivors. Leukocytosis was the main abnormality (54.8%) detected on diagnosis, from the leukocyte count. During the evolution, normal leukocyte count was associated with survival (p<0.01) and leukocytosis with mortality (p<0.05). In conclusion, mortality was associated with nondetection of the pathogen, leukocytosis during the evolution of the sepsis and inappropriate or empirical use of antimicrobials. Evidence-based treatment that is directed towards modifiable risk factors might improve the prognosis for sepsis patients.

Tumor sections from nonneoplastic (n = 15), benign (n = 28), and malignant ovarian tumors (n = 20) were obtained from 63 women. Immunohistochemistry of the tumor sections demonstrated that inducible nitric oxide synthase (iNOS) expression was increased in ovarian cancer samples compared to nonneoplastic or benign tumor samples. Using the Griess method, nitric oxide (NO) metabolite levels were also found to be elevated in malignant tumor samples compared to benign tumor samples (P < .05). For stage I ovarian cancer, intracystic NO levels >80 microM were more frequent than NO levels <80 microM, and iNOS expression in well-differentiated carcinomas was greater than in moderately/poorly differentiated carcinomas (P < .05). These data suggest an important role for NO in ovarian carcinogenesis.

Purpose: To verify the relationship between clinical variables and tumor stage in breast cancer., Methods: This retrospective study (1998 to 2001) analyzed data of 176 women with breast cancer attending a university hospital. Patients were divided into groups according to the clinicopathological variables studied., Results: The disease had a similar frequency at age under 50 years (44.3%) or above (55.7%) 50 years. Stage II was more frequent. Most patients were white (69.9%), non-smokers (69.3%) and were not using oral contraceptives (71%). Stages 0-II were mainly detected in the white (74.8%) vs non-white (60.4%) group. Monthly breast self-exams were performed by 62.5% of women, in which earlier stages (0, I) were more frequently detected than in those who did not perform self-exams (27.3% vs 12.1%, p = 0.01)., Conclusion: Breast cancer occurred mainly in white women in Stage II, and with similar frequency at age under or over 50 years. Breast self-exam was associated with early detection of the disease.

The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. alpha-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 microg per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced the l-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from approximately 80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.

Neutrophil migration is a key event in the inflammatory response of any origin, and neutrophils may present antitumor activity. We investigated the number and function of circulating neutrophils obtained from patients with cervical neoplasia at different stages. Patients with preinvasive (cervical intraepithelial neoplasia, CIN3, n= 6) or microinvasive ([MICRO] stage IA1, n= 4) neoplasia were evaluated together as CIN/MICRO group (n= 10), while patients at stages II-IV were evaluated as invasive group (INV, n= 12). Healthy women served as controls (n= 15). For patients, analysis of leukogram on diagnosis showed a significant elevated neutrophil count in INV group compared with that in CIN/MICRO group. A neutrophil/lymphocyte ratio >/=5 was observed in 67% patients from INV group compared with only 10% from CIN/MICRO group. Neutrophil migration, assayed in a microchemotaxis chamber in response to the chemoattractants (10(-7) M) N-formyl-l-methionyl-l-leucyl-l-phenylalanine, leukotriene B(4), or interleukin-8, was reduced in INV group than in controls or CIN/MICRO group. Surgical treatment in randomly selected patients from CIN/MICRO group (four CIN, one MICRO) increased neutrophil migration to all chemoattractants compared with time on diagnosis. The serum levels of nitric oxide (NO) metabolites, assayed by the Griess reaction, were higher in patients (n= 19) than in controls (n= 15), without differences related to tumor stage, but were reduced in patients after surgery compared with pretreatment (n= 10). Taken together, the results suggest that neutrophils play a role in the host response in cervical cancer. Soluble circulating mediators released by tumor cells, such as NO, could interfere early in the capacity of neutrophils to migrate, thus impairing host immune response.

Purpose: To investigate the effects of different conditions used during cardiopulmonary bypass (CPB) surgery on accompanying production of cytokine and nitric oxide (NO)., Methods: Patients undergoing CPB for the first time were prospectively enrolled and divided into two groups according to CPB parameters performed: i) normothermia (36.5-37 degrees C) with blood cardioplegia (NB group, n=10) and ii) hypothermia (29-31 degrees C) with crystalloid cardioplegia (HC group, n=10). Plasma samples obtained following intubation (baseline), during (5 and 30 min) and after (4 and 24 h) CPB were assayed for cytokines (ELISA) and NO metabolites (Griess reaction)., Results: Peak concentrations of interleukin (IL)-6 and IL-8 were reached at 4 h post CPB in both groups, but in the HC group those levels increased earlier and persisted for longer (24 h) compared to baseline (P < 0.05). IL-10 levels also increased at 4 h compared to baseline, but only significantly so in the HC group. NO metabolites were reduced in HC group at all time points compared to baseline (P < 0.05), while no significant differences were detected in the NB group., Conclusion: The association between increased systemic levels of cytokines and reduced NO production in the HC group suggests that different myocardial protection and/or perfusion temperature used during CPB may contribute to the extent of inflammatory response.

The deregulation of inflammatory response during sepsis seems to reflect the overproduction of mediators, which suppress leukocyte functions. We investigated the intracellular mechanisms underlying the inability of neutrophils from severe septic patients to migrate toward chemoattractants. Patients with sepsis (52) and 15 volunteers were prospectively enrolled. Patients presented increased circulating levels of tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-8, and IL-10. Patients showed reduced neutrophil chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or IL-8. No difference in the transcription or expression of the IL-8 receptor, CXCR1, was detected in neutrophils from controls and patients. However, septic neutrophils failed to increase tyrosine phosphorylation and actin polymerization in response to IL-8 or LTB4. In contrast, septic neutrophils, similar to controls, showed phagocytic activity that induced actin polymerization and augmented phosphotyrosine content. Treatment of control neutrophils with cytokines and lipopolysaccharide (LPS) to mimic endogenous septic environment inhibited actin polymerization and tyrosine phosphorylation in response to IL-8 or LTB4. High expression of G protein-coupled receptor kinase 2 (GRK2) and GRK5 was detected in septic neutrophils and control cells treated with cytokines plus LPS. Data suggest that endogenous mediators produced during sepsis might continually activate circulating neutrophils, leading to GRK activation, which may induce neutrophil desensitization to chemoattractants.

Sepsis and septic shock continue to be a major cause of morbidity and mortality in critically ill patients. During the onset of sepsis, several inflammatory mediators, including cytokines, chemokines and nitric oxide are released systemically and mediate most of the pathophysiological events present in sepsis and septic shock, such as cardiovascular dysfunction and target-organ lesions. Polymorphonuclear leukocytes are critical effector cells during the inflammatory process and their migration to the infection focus is extremely important for the local control of bacterial growth and consequently for the prevention of bacterial dissemination. In experimental models and in human sepsis a profound failure of neutrophil migration to the infection focus is observed. It seems that the failure of neutrophil migration is dependent on toll-like receptor 4 (TLR4) and mediated by cytokines and chemokines, which induce the production of nitric oxide that inhibits neutrophil adhesion to venular endothelium and also the neutrophil chemotactic ability.

Neutrophil migration is a key host event against infection. Chemotherapy may alter neutrophil function and favor increased risk of infection. Herein, we investigated the effect of chemotherapy on the migration capacity of circulating neutrophils obtained from breast cancer patients and mechanisms involved in this event. Breast cancer women (n=23) at disease stage I-III and healthy control women (n=25) were prospectively enrolled. No differences in the in vitro migratory responses towards the chemotactic stimuli N-formyl- L-methionyl- L-leucyl- L-phenylalanine (fMLP), leukotriene B(4) (LTB(4)) and interleukin (IL)-8 were observed in purified neutrophils from controls and patients, in a microchemotaxis chamber assay. However, the migration capacity evaluated upon chemotherapy (5-fluoruracil, adriamycin and cyclophosphamide, 21-day intervals between cycles, total leukocyte count >/=2,000/mm(3)), on the day immediately before the beginning of the sixth cycle, showed that patient neutrophils (n=14) failed to migrate in response to fMLP compared to response observed upon diagnosis. Considering patients (n=8) with documented bacterial infection between cycles, the number of migrated neutrophils (mean+/-SD) compared to response at diagnosis was markedly reduced upon chemotherapy to either fMLP (30.1+/-8.26 vs. 2.81+/-1.28) or LTB(4) (15.72+/-4.8 vs. 2.8+/-1.64) stimuli respectively. Treatment of control neutrophils with sera of chemotherapy-treated patients with infective episodes, to test for the presence of circulating immunosuppressive factors, significantly reduced the migratory capacity of healthy neutrophils to fMLP, LTB(4) and IL-8, in a dose-dependent way. But no significant differences were found in the serum levels of nitric oxide (NO) metabolites, tumor necrosis factor (TNF)-alpha, IL-6, IL-8 and IL-10 collected at the same time as the collection of blood for neutrophil migration experiments. In conclusion, breast cancer patients showed suppressed neutrophil migratory response upon chemotherapy, accompanied by bacterial infection episodes. Circulating factors are involved, at least partially, in the inhibitory mechanism on neutrophil migration.

Sepsis is a systemic inflammatory response commonly caused by bacterial infection. We demonstrated that the outcome of sepsis induced by cecal ligation and puncture (CLP) correlates with the severity of the neutrophil migration failure towards infectious focus. Failure appears to be due to a decrease in the rolling and adhesion of neutrophil to endothelium cells. It seems that neutrophil migration impairment is mediated by the circulating inflammatory cytokines, such as TNF-alpha and IL-8, which induce the nitric oxide (NO) production systemically. It is supported by the fact that intravenous administration of these cytokines reduces the neutrophil migration induced by different inflammatory stimuli, and in severe sepsis the circulating concentrations of the cytokines and chemokines are significantly increased. Moreover, the neutrophil migration failure and the reduction in the rolling/adhesion were not observed in iNOS-/- mice and, aminoguanidine prevented this event. We also demonstrated that the failure of neutrophil migration is a Toll-4 receptor (TLR4) dependent mechanism, since it was not observed in TLR4 deficient mice. Furthermore, it was also observed that circulating neutrophils obtained from septic patients present failure of neutrophil chemotaxis toward fMLP, IL-8, and LTB4 and an increased in sera concentrations of NO3 and cytokines. In conclusion, we demonstrated that, in sepsis, failure of neutrophil migration is critical for the outcome and that NO is involved in the process.

Aims and Background: Cells and soluble mediators obtained from tumor effusions are useful in evaluating the tumor microenvironment. Our aim was to examine cytologically and to quantify the leukocyte infiltrate, nitric oxide, cytokines and tumor markers in the intracystic fluid from patients with a cystic adnexal mass, for a possible differentiation between benign and malignant findings., Methods and Study Design: Sixty-six women who had their cystic fluids collected were prospectively divided into benign tumor (22, 33.3%), malignant tumor (10, 15.2%) or other gynecological alterations (34, 51.5%). Cytology, total and differential leukocyte counts were determined by light microscopy. Tumor markers, cytokines and nitric oxide were assayed in the supernatants using the Immulite system, ELISA and Griess reaction, respectively., Results: The sensitivity and specificity of the cytological analysis was 66.7% and 97.7%, respectively. The levels of CA 19.9, CA 15.3, alpha-fetoprotein, carcinoembryonic antigen, progesterone and beta-HCG were significantly higher in the benign and/or malignant group than in the other gynecological alterations. Also, the local concentrations of CA 15.3 and beta-HCG were significantly higher in malignant than in benign tumors. In malignant tumors, increased leukocyte counts and higher concentrations of IL-6, IL-10 and nitric oxide were detected than in benign tumors or other gynecological alterations., Conclusions: In malignant tumors, the microenvironment could be differentiated from benign tumors or other gynecological alterations by cystic fluid analysis.

A 25-year-old white woman with a vaginal invasive epidermal carcinoma was treated with interferon-alpha 2b (3,000,000 U intralesional, every two days for a total of 18 applications). Follow-up with Papanicolaou and colposcopy showed complete regression of the vaginal lesion, After three years the patient got pregnant. The prenatal course was uneventful and a vaginal delivery was performed. She is currently in follow-up without any signs of recurrence.

Purpose of Investigation: To identify parameters for the diagnosis of ovarian neoplasia using ultrasonography (US) and serum tumor marker (TM: CA125, CA19.9, CA15.3, AFP, CEA and estradiol) assay., Methods: Prospective study which included 373 women with increased ovarian volume (> 18 cm3 in premenopause and > 8 cm3 in postmenopause). US criteria (> or = 1) for surgery were: persistent (> 4 months) or increased cyst, cysts with > 1 thick septum or > or = 2 thin septa, cyst diameter > or = 7 cm, vegetation, calcification or cystic predominance (> 50%), solid tumor (> 50%). Doppler with a resistance index (RI) < 0.4 was considered abnormal., Results: Laparotomy was performed in 164 (44%) patients with 66 (40.2%) benign neoplasias and 19 (11.6%) malignant cases (73.6% at Stage I or II). Two hundred and nine patients were maintained on clinical follow-up. The sensitivity for neoplasia and malignant neoplasia was, respectively, for RI: 17 and 63.6 and RI plus TM: 53.1 and 90.9., Conclusion: Ultrasound criteria and TM assay were indicated for the diagnosis of ovarian neoplasia.

Objective: To investigate the in vitro chemotactic function of neutrophils obtained from patients with sepsis., Design: Prospective study in which purified neutrophils obtained from septic patients and nonseptic control volunteers were assayed for chemotactic function induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and leukotriene B4. The sera nitrate concentrations also were quantified., Setting: University hospital., Patients: Twenty patients with sepsis caused by different infectious foci., Interventions: Routine blood tests, blood or other site cultures, blood collection for neutrophil purification sera collection for nitrate assay., Measurements and Main Results: Neutrophils from septic patients exhibited significantly less chemotactic activity than neutrophils obtained from healthy volunteers, in response to FMLP (93.4 +/- 6.6 vs. 51 +/- 8.3 migrated neutrophils) and leukotriene B4 (90.2 +/- 10 vs. 42.4 +/- 11.6 migrated neutrophils) stimuli, in a microchemotaxis chamber assay. The impaired chemotaxis occurred mainly in neutrophils from nonsurvivor patients. The extent of neutrophil chemotaxis inhibition (survivor/nonsurvivor) was 33.43%/61.67% and 43.4%/86.98%, in response to FMLP and leukotriene B4, respectively. Increased serum nitrate (micromoles of NO2 + NO3) concentrations were detected in septic patients, compared with controls, but no differences were found between survivor (91.84 +/- 14.12) and nonsurvivor (102.6 +/- 17.36) groups., Conclusions: Septic patients present suppressed neutrophil chemotactic responses to FMLP and leukotriene B4 stimuli compared with healthy controls. This is accompanied by increased serum concentrations of nitrate. The impairment of neutrophil chemotaxis was observed mainly in the cells obtained from nonsurvivor patients and may thus be an additional factor contributing to disease outcome.

With the aim of studying the frequency of Chagas' disease among sufferers of uterine leiomyoma, we analyzed women older than 35 years who underwent surgery and presented with leiomyoma on anatomicopathological examination. The diagnosis of Chagas infection was based on positivity to at least two of three serological tests: enzyme-linked immunosorbent assay, passive hemagglutination, and immunofluorescence. The study was case controlled, matching for age, skin color, and parity. The control group consisted of women undergoing surgery for other benign gynecological alterations. During this period, 118 women presented with uterine leiomyoma, 27.1% of whom were serologically positive for Chagas' disease versus 16.1% of the controls (P < 0.05). Matching by skin color and parity showed that 40% of the white multiparous women with uterine leiomyoma had Chagas' disease versus 10% of the controls (P < 0.05). We concluded that there appears to be an association between Chagas' disease and uterine leiomyoma.

To investigate the role of NO in the inhibition of neutrophil migration by circulating endotoxin, mice were pretreated with NO synthase inhibitors or with a free radical scavenger (D-penicillamine), before intravenous LPS injection. LPS dose-dependently inhibited the thioglycollate-induced neutrophil migration into the peritoneal cavities. Aminoguanidine, a selective inducible NO synthase inhibitor, abolished the inhibition of neutrophil migration and the increase in serum nitrate levels induced by a nonlethal dose of LPS. During lethal endotoxemia aminoguanidine partially abolished the neutrophil migration inhibition. Additionally, D-penicillamine prevented the inhibition of neutrophil migration caused by LPS. However, Nitro-L-Arginine, a selective constitutive NO synthase inhibitor, did not prevent neutrophil migration inhibition. Aminoguanidine treatment did not affect the systemic increased levels of TNF-alpha, IL-1beta, and IL-10, suggesting that NO is the final mediator involved in the inhibition of neutrophil migration. Our results suggest that NO released by the inducible NO synthase mediates the inhibition of neutrophil migration mediated by circulating LPS.

Aims: This brief review focuses on the current understanding of the complex relationship of tumor-associated mononuclear cells (TAMs) with neoplastic cells, summarizing their immunological efficiency, cytokine profile and production of nitric oxide (NO) in the tumor microenvironment, with current insights on how this might affect tumor growth., Data Source: Data was obtained through Medline from articles indexed during the last 10 years. The main key words used in the research were: cancer, ovarian cancer, cytokine, nitric oxide (NO), mononuclear cell, lymphocyte, macrophage. SELECTION OF STUDIES AND DATA COLLECTION: 30 studies were reviewed, which contained data regarding the production of cytokines and NO by TAMs or malignant cells, and tried to establish a correlation between these mediators and tumor growth, especially in ovarian carcinoma., Data Summary: TAMs consist mainly of macrophages and T lymphocytes which present lower proliferative indices and cytotoxicity compared to autologous blood monocytes, although they are able to release various cytokines. The profile of cytokine expression could help to explain both the immunological impairment observed in patients with advanced carcinoma diseases and the potential of TAMs to exert antitumor activity, which makes these cells an attractive target for therapeutic intervention. NO is also produced in the tumor microenvironment. Several reports in animals suggest a tumoricidal role for NO, but in human tumors its role has not been well-established and may change during tumor progression.

1. The i.v. administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site. 2. Pretreatment of mice with the NO synthase antagonist, NG-monomethyl-L-arginine (L-NMMA, 15-60 mg kg(-1)), but not the inactive enantiomer D-NMMA (30 mg kg(-1)), prevented in a dose-dependent manner the TNF-alpha, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities. 3. Treatment of the neutrophils with TNFalpha (10(-7) M), IL-8 (10(-7) M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50-200 microM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-alpha or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis. 4. Preincubating the neutrophils with L-NMMA (200 microM) significantly increased the TNF-alpha (10(-7) M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10(-7) M)-mediated adhesion. 5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-alpha and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-alpha-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.

There is controversy regarding the evidence for the production of nitric oxide (NO) by neutrophils (PMNs). The present study investigates NO production, as assessed by the biosynthesis of the end products, nitrite and nitrate, in the pellets and supernatants of rat and mouse peripheral blood neutrophils obtained during endotoxemia and of peritoneal carrageenin-elicited PMNs stimulated in vitro with E. coli lipopolysaccharide (LPS). We also investigated the induction of NO synthase by rat and mouse peritoneal cells. The intraperitoneal (ip) administration of LPS to mice (10 mg/kg) and rats (5 mg/kg) significantly increased plasma nitrate concentration by six and 23-fold, respectively. In vivo pretreatment with L-NGmonomethyl arginine (L-NMMA) significantly inhibited this production. Compared to animals injected with PBS, the cell pellets of blood PMNs obtained from mice, but not rats, 2 or 6 h after LPS administration produced significant amounts of nitrite (14 +/- 3 and 18 +/- 2 nmol/mg protein, respectively). Little or no nitrite was found in the incubating medium. In contrast, 6 h after a carrageenin challenge (700 micrograms) peritoneal neutrophils obtained from rats, but not mice, released high concentrations of nitrite into the supernatant during a 24-h period of incubation (34 +/- 0.8 microM). The nitrite concentration of the pellet of these cells was negligible. In contrast to the lack of increase in the amount of nitrite released into the supernatants, the in vitro stimulation of rat PMNs with LPS (10 micrograms/ml) for 24 h did increase intracellular nitrite concentration (from 0.8 +/- 0.07 to 8 +/- 0.3 nmol/mg protein). In mouse PMNs, LPS treatment caused only a small release of nitrite into the incubation medium (14 +/- 1 microM). There was no significant change in nitrite concentration in the cell pellets. These data suggest that rat and mouse neutrophils differ in their ability to produce nitric oxide following stimulation with endotoxin.

In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-alpha and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal(-)) and bound (D-gal(+)) fractions, with MNCF being found in the D-gal(+) fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal(-) fraction. Furthermore, the incubation of the D-gal(-) fraction with anti-TNF-alpha plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal(+) activity. Overall, these results suggest that the D-gal(-) inhibitory effect is partially mediated by TNF-alpha and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal(+) fraction.

1. A neutrophil recruitment inhibitory factor (NRIF) recovered from the crude supernatant of lipopolysaccharide (LPS)-stimulated macrophages inhibited neutrophil migration following both intratracheal and intravenous administration of LPS, but did not alter the pattern of leukopenia/leucocytosis induced by intravenous LPS. 2. The correlation between airway infiltration by inflammatory cells and hyperreactivity in lungs from actively sensitized and challenged guinea-pigs was investigated by use of NRIF. 3. Increased eosinophil counts were found in the bronchoalveolar lavage fluid from guinea-pigs sensitized with 10 micrograms ovalbumin and challenged at day 14 by the intranasal administration of the antigen. The increase was evident 5 h after challenge and persisted at 24 h. Neutrophil numbers were also increased at this time. Pretreatment with NRIF suppressed the leucocyte increase in the bronchoalveolar lavage fluid. 4. Bronchoconstriction and histamine release induced by 3 ng PAF injected into the isolated lungs were increased in challenged guinea-pigs as compared to sensitized but unchallenged controls. Pretreatment of the animals with NRIF did not interfere with this response, but significantly reduced the bronchoconstriction induced by ovalbumin injection. 5. Even though the increased number of inflammatory cells in bronchoalveolar lavage and airway hyperresponsiveness were concomitant, NRIF inhibited cellular infiltration but failed to alter airway hyperreactivity to PAF, demonstrating that these events may occur independently. Conversely, the inhibition of antigen-induced bronchoconstriction by NRIF suggests that this response is dependent upon the emigration of granulocytes.

Inhibitory effect upon neutrophil migration to the inflammatory focus was previously detected in the cell-free incubation fluid of lipopolysaccharide (LPS)-stimulated macrophage monolayers. In the present study we showed that the neutrophil recruitment inhibitory activity from this supernatant was mainly detected in a fraction (P2) obtained by gel filtration chromatography on Sephacryl S-300. P2 fraction was able to inhibit 'in vivo' neutrophil emigration induced by different inflammatory stimuli, but it did not affect 'in vitro' neutrophil chemotaxis induced by FMLP. When injected intravenously, P2 inhibited oedema induced by carrageenin or immunological stimulus but not the oedema induced by dextran, thus affecting cell-dependent inflammatory responses. It was observed that P2 also induced neutrophil migration when injected locally in peritoneal cavities. This activity was significantly reduced by pretreatment of the animals with dexamethasone. Cytokines, such as IL-8 and TNF-alpha that are known to exhibit inhibitory effect upon neutrophil migration, were not detected in P2 fraction by highly sensitive assays. Overall the results suggest the existence of a novel cytokine exhibiting 'in vivo' neutrophil inhibitory activity, referred as NRIF.