Multiresistant Acinetobacter baumannii causing malignant otitis externa: a case report.

Abstract: Malignant otitis externa is an infection of the skin and soft tissue of the ear canal, spreading to the nearby structures. It causes severe otalgia and otorrhea, and can lead to ominous consequences such as cranial nerve damage and meningitis. The main etiologic agent is Pseudomonas aeruginosa and treatment relies on broad-spectrum intravenous antibiotics. We report a rare case of a woman suffering from Malignant otitis externa caused by Acinetobacter baumannii and requiring the use of colistin.

Evaluation of Discomfort in Nasopharyngeal Swab Specimen Collection for SARS-CoV-2 Diagnosis.

BACKGROUND: The rapid spread of COVID-19 worldwide has impo-sed the need to identify a test that quickly recognizes affected subjects, both symptomatic and asymptomatic. The most reliable option has been proven to be the RT-PCR, which allows to detect virus RNA on a specimen from a high viral load site, such as nasopharynx. Nasopha-ryngeal sample collection is possible by means of a nasopharyngeal swab (NPS) and is a practical and relatively non-invasive technique, but rather bothersome for the recipient. AIM: The aim of the present study is to evaluate the discomfort evoked during NPS. MATERIALS AND METHODS: We surveyed 429 patients receiving NPS before hospitalization or other procedures non related to COVID-19. For each one we noted the discomfort level felt during the swab using a 11-point numeric rating scale (NRS) for pain and the total time needed for the procedure to be taken. Sex, age, smoking status and positive history of previous swab have been taken into account. RESULTS: We found that, among the variables, sex had a statistically significant impact on the perceived discomfort of nasal swab, with females experiencing slightly more discomfort. CONCLUSIONS: NPS is largely a none-to-minimum discomfort in-ducing procedure. The differences in perceived discomfort could be explained based on anatomical features, and should remark the need for a tailored and anatomy-oriented approach in each patient.

Intervertebral disc and endplate cells response to IL-1ß inflammatory cell priming and identification of molecular targets of tissue degeneration.

Inflammation represents an important factor leading to metabolic imbalance within the intervertebral disc (IVD), conducive to degenerative changes. Therefore, a thorough knowledge of the IVD and endplate (EP) cell behaviour in such pathological environments is essential when designing regenerative therapeutic strategies. The present study aimed at assessing the molecular response of the IVD constitutive nucleus pulposus (NPCs)-, annulus fibrosus (AFCs)- and endplate (EPCs)-derived cells to interleukin (IL)-1ß treatment, through large-scale, high-throughput microarray and protein analysis, identifying the differentially expressed genes and released proteins. Overall, the inflammatory stimulus downregulated stemness genes while upregulating pro-inflammatory, pro-angiogenic and catabolic genes, including matrix metalloproteases, which were not balanced by a concomitant upregulation of their inhibitors. Upregulation of anti-inflammatory and anabolic tumour necrosis factor inducible gene 6 protein (TNFAIP6), of IL-1 receptor antagonist (IL-1Ra) (at gene and protein levels) and of trophic insulin-like growth factor 1 (IGF1) was also observed in all cell types; IGF1 particularly in AFCs. An overall inhibitory effect of tumour necrosis factor alpha (TNFα) signal was observed in all cell types; however, EPCs showed the strongest anti-inflammatory behaviour. AFCs and EPCs shared the ability to limit the activation of the signalling mediated by specific chemokines. AFCs showed a slightly senescent attitude, with a downregulation of genes related to DNA repair or pro-mitosis. Results allowed for the identification of specific molecular targets in IVD and EP cells that respond to an inflammatory environment. Such targets can be either silenced (when pathological targets) or stimulated to counteract the inflammation.

Acinic cell carcinoma of the parotid gland: from pathogenesis to management: a literature review.

PURPOSE: Acinic cell carcinoma (ACCs) is uncommon malignant epithelial neoplasm of the salivary glands; the most common presentation is a well-defined painless solid mass. Diagnosis of ACCs is frequently complicated, due to its similarity with benign tumors. METHODS: A review of the literature available on ACCs was carried out. Studies were sourced from PubMed with searching of relevant headings and sub-headings and cross-referencing. RESULTS: There are no clear characteristics of ACCs found on CT, MRI and ultrasound imaging. The management of the ACC, a rare malignancy of the parotid gland, is often difficult and controversial. Radical surgery is the best treatment option. The role of radiotherapy remains controversial: the precise indications and oncologic effects of adjuvant radiotherapy in ACC of the parotid gland are not well known. There is insufficient literature regarding the chemotherapy for metastatic ACC. CONCLUSION: Knowledge about ACC, a rare malignancy of parotid gland, has changed over the past few decades. More clinical randomized works would be needed, both to assess the real effectiveness of radio and chemotherapy and to have an unanimous consensus about their indications.

BRCA1 and p53 regulate critical prostate cancer pathways.

BACKGROUND: Loss or mutations of the BRCA1 gene are associated with increased risk of breast and ovarian cancers and with prostate cancer (PCa) aggressiveness. Previously, we identified GADD153 as a target of BRCA1 protein, which increases doxorubicin sensitivity in human p53 -/- PCa cells (PC3). Considering that p53 is a crucial target in cancer therapy, in this work we investigated p53 role in the regulation of transcription of GADD153. METHODS: We performed reverse transcription quantitative PCR (RT-qPCR), western blot and luciferase assays to analyze GADD153 and/or BRCA1 expression in response to ultraviolet or doxorubicin exposure in PC3 p53 stable-transfected cells and LNCaP (p53+/+) cells. BRCA1 protein recruitment to GADD153 promoter was studied by chromatin immunoprecipitation-qPCR. To assess expression of BRCA1 and/or p53 target genes, we used a panel of stable-transfected PCa cell lines. We finally analyzed these genes in vivo using BRCA1-depleted PCa xenograft models. RESULTS: We found that GADD153 was highly induced by doxorubicin in PC3 cells; however, this response was totally abolished in LNCaP (p53wt) and in p53-restituted PC3 cells. Furthermore, BRCA1 protein associates to GADD153 promoter after DNA damage in the presence of p53. Additionally, we demonstrated that BRCA1 and/or p53 modulate genes involved in DNA damage and cell cycle regulation (cyclin D1, BLM, BRCA2, DDB2, p21(WAF1/CIP1), H3F3B, GADD153, GADD45A, FEN1, CCNB2), EMT (E-cadherin, ß-catenin, vimentin, fibronectin, slug, snail) and Hedgehog pathways (SHH, IHH, DHH, Gli1, PATCH1). Furthermore, xenograft studies demonstrated that BRCA1 knockdown in PC3 cells increased tumor growth and modulated these genes in vivo. CONCLUSIONS: Although BRCA1 induces GADD153 in a p53 independent manner, p53 abolished GADD153 induction in response to DNA damage. In addition, several important PCa targets are modulated by BRCA1 and p53. Altogether, these data might be important to understand the therapy response of PCa patients.

Multifunctional CD4⁺ T cells in patients with American cutaneous leishmaniasis.

Leishmaniasis is a group of important parasitic diseases affecting millions worldwide. To understand more clearly the quality of T helper type 1 (Th1) response stimulated after Leishmania infection, we applied a multiparametric flow cytometry protocol to evaluate multifunctional T cells induced by crude antigen extracts obtained from promastigotes of Leishmania braziliensis (LbAg) and Leishmania amazonensis (LaAg) in peripheral blood mononuclear cells from healed cutaneous leishmaniasis patients. Although no significant difference was detected in the percentage of total interferon (IFN)-γ-producing CD4(+) T cells induced by both antigens, multiparametric flow cytometry analysis revealed clear differences in the quality of Th1 responses. LbAg induced an important proportion of multifunctional CD4(+) T cells (28% of the total Th1 response evaluated), whereas LaAg induced predominantly single-positive cells (68%), and 57% of those were IFN-γ single-positives. Multifunctional CD4(+) T cells showed the highest mean fluorescence intensity (MFI) for the three Th1 cytokines assessed and MFIs for IFN-γ and interleukin-2 from those cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulation. These major differences observed in the generation of multifunctional CD4(+) T cells suggest that the quality of the Th1 response induced by L. amazonensis antigens can be involved in the mechanisms responsible for the high susceptibility observed in L. amazonensis-infected individuals. Ultimately, our results call attention to the importance of studying a Th1 response regarding its quality, not just its magnitude, and indicate that this kind of evaluation might help understanding of the complex and diverse immunopathogenesis of American tegumentary leishmaniasis.

Upregulation of miR-21 by Ras in vivo and its role in tumor growth.

miR-21 is a microRNA (miRNA) frequently overexpressed in human cancers. Here we show that miR-21 is upregulated both in vitro and in vivo by oncogenic Ras, thus linking this miRNA to one of the most frequently activated oncogenes in human cancers. Ras regulation of miR-21 occurs with a delayed kinetic and requires at least two Ras downstream pathways. A screen of human thyroid cancers and non-small-cell lung cancers for the expression of miR-21 reveals that it is overexpressed mainly in anaplastic thyroid carcinomas, the most aggressive form of thyroid cancer, whereas in lung its overexpression appears to be inversely correlated with tumor progression. We also show that a LNA directed against miR-21 slows down tumor growth in mice. Consistently, a search for mRNAs downregulated by miR-21 shows an enrichment for mRNAs encoding cell cycle checkpoints regulators, suggesting an important role for miR-21 in oncogenic Ras-induced cell proliferation.

Modulation of the catalytic activity of free and immobilized peroxidase by extremely low frequency electromagnetic fields: dependence on frequency.

A study of the influence of electromagnetic fields (EMF) of various frequencies, from 50 up to 400 Hz, on the catalytic activity of soluble and insoluble horseradish peroxidase (POD) was carried out. To simulate the conditions in which the enzyme operates in vivo, the POD was immobilized by entrapment on a gelatin membrane or by covalent attachment on a nylon graft membrane. The rate of inactivation of the soluble POD was found to exhibit positive and negative interactions with the 1 mT applied magnetic field, with an optimum positive effect at 130 Hz. The immobilized PODs, on the contrary, do not exhibit negative interactions, but show a maximum positive interaction at 150 Hz when entrapped and at 170 Hz when covalently attached. At 50 Hz and at frequencies higher than 250 Hz no effects were observed with insoluble POD. The optimum frequency of positive interaction between the EMF and the catalytic activity of the insoluble enzymes is shifted with respect to that of the soluble enzymes towards higher frequencies, the size of the shifts being dependent on the intensity of the physical forces involved in the immobilization process.

In vitro studies of the influence of ELF electromagnetic fields on the activity of soluble and insoluble peroxidase.

The influence of an extremely low frequency (ELF) magnetic field (50 Hz and 1 mT, EMF) on the activity of a soluble and insoluble horseradish peroxidase (E.C. 1.11.17) has been studied as a function of time. Insoluble derivatives were obtained by enzyme entrapment into two different gelatin membranes or by covalent attachment of the enzyme on two nylon membranes, differently preactivated. Results have shown that the field affects the inactivation rate of the soluble enzyme, while no effects are observed with insoluble derivatives. Since in vivo enzymes are immobilised into the biomembrane bilayer or entrapped into the cytoplasmic mixture, one might speculate that our experimental conditions do not reflect the catalytic activity of the enzymes in vivo.

[Case report: Acro-necrosis of the upper limbs caused by gemcitabine therapy]. / Acronecrosi degli arti superiori da gemcitabina: segnalazione di un caso clinico.

INTRODUCTION: Even if infrequent, a digital necrosis after chemotherapy can occur in cancer patients. The gemcitabine is generally well tolerate; the cutaneous toxic ulcerations only in 0.3% of the cases induces the suspension of the treatment. CLINICAL CASE: A 70 year old patient, female, with a bladder cancer, after a trans-urethral resection, is submitted to adjuvant chemotherapy with Gemcitabine 1700 mg (total dose/die), with administration in the days 1st and 8th, while in the 15th day was not effected because, to distance of 3-4 days from the second administration, appear paresthesies of the fingers of the hands, together with Raynaud type phenomenon, 38-39 degrees C intermittent fever, digital necrosis and fingertips gangrene. Laboratory: (Normal): RF; AutoAb: AMA, ASMA, APCA, anti-DNA; ENA; lupus anti-coagulant; Ab-anti-cardiolipin; C3-C4, CIC; homocysteine, anti-thrombin, protein C, protein S, mutation of the factor V of Leiden, plasminogen, alfa 2-antiplasmin. (Altered): Auto-antibody: ANA (on Hep-2): positive (title 1/160, speckled pattern), cryoglobulin positive, ESR 29; Instrumental examinations: Superior Limbs Angiograpy: Occlusion of the digital arteries proper of 2nd, 3rd and 4th finger of the hands. Electromyography Inferior Arts: normal. Superior Arts: bilateral suffering of the median nerve at the carpal tunnel. Biopsy of the hand cutis: Hyperkeratosis, acanthosis and papillomatosis of the skin. Arterial vases with signs of endothelioangiitis and aspecific inflammation. CONCLUSIONS: Even if acronecrosis of the superior limbs is a rare effect of the gemcitabine, we would recommend particular caution in the administration of this drug in patient with known autoimmune disorders.

Body composition analysis and changes in airways function in obese adults after hypocaloric diet.

STUDY OBJECTIVES: To determine the relationship between weight-loss and pulmonary function indexes, focusing on forced expiratory flows (ie, FEV(1), forced expiratory flow at 50% of vital capacity [FEF(50)], forced expiratory flow at 75% of vital capacity, and forced expiratory flow at 25 to 75% of vital capacity [FEF(25--75)]). Specifically, to determine the effect of losses in total and segmental fat mass (FM) and of modifications in lean body mass, after restricted hypocaloric diet, on pulmonary function among obese adults. DESIGN: Cross-sectional, observational. SETTINGS: Human Physiology Division, Faculty of Medicine and Surgery, "Tor Vergata" University, Rome, Italy. PATIENTS: Thirty obese adults (mean [+/- SD] baseline body mass index [BMI], 32.25 +/- 3.99 kg/m(2)), without significant obstructive airway disease, were selected from among participants in a weight-loss program. MEASUREMENTS AND RESULTS: Anthropometric, body composition (BC), and respiratory parameters of all participants were measured before and after weight loss. Total and segmental lean body and FM were obtained by dual-energy x-ray absorptiometry. Dynamic spirometric tests and maximum voluntary ventilation (MVV) were performed. The BC parameters (ie, body weight [BW], BMI, the sum skinfold thicknesses, thoracic inhalation circumference, thoracic expiration circumference, total FM, and trunk FM [FMtrunk]) were significantly decreased (p < or = .0001) after a hypocaloric diet. The mean vital capacity, FEV(1), FEF(50), FEF(25-75), expiratory reserve volume, and MVV significantly increased (p < or = 0.05) with weight loss. The correlation coefficient for Delta FEF(25--75) (r = 0.20) was numerically higher than Delta FEF(50) and Delta FEV(1) (r = 0.14 and r = 0.08, respectively) for the BW loss. Moreover, the correlation coefficient for Delta FEF(25--75) (r = 0.45) was significantly higher (p < or = 0.02) than those for Delta FEF(50) and Delta FEV(1) (r = 0.38 and r = 0.15, respectively) for FMtrunk loss. CONCLUSIONS: This study shows that a decrease in total and upper body fat obtained by restricted diet was not accompanied by a decrease in ventilatory muscle mass. FMtrunk loss was found to have improved airflow limitation, which can be correlated to peripheral airways function.

Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

Class A helix-loop-helix proteins are positive regulators of several cyclin-dependent kinase inhibitors' promoter activity and negatively affect cell growth.

The class A of basic helix-loop-helix (bHLH) proteins are ubiquitously expressed transcription factors playing a pivotal role in the regulation of cell growth and differentiation. We determined that enforced expression of all four different mammalian members of this family, E12, E47, E2-2, and HEB, suppresses the cell colony-forming efficiency of several cell lines. To gain insights into the mechanisms by which class A bHLH factors affect cell growth, we have investigated their role in the transcriptional regulation of cyclin-dependent kinase inhibitors. We found that p21CIP1/ WAF1, p15INK4B, and p16INK4B promoter sequences contain E-boxes that render these genes competent for class A bHLH-mediated transcriptional activation and Id-mediated repression. The mechanism underlying the class A bHLH-mediated inhibition of cell growth does not involve an arrest of G1 progression in 293T cells. In fact, contrary to what has been found in 3T3 NIH fibroblasts, we found that enhanced expression of class A bHLH proteins led to a decreased proliferation rate by promoting cell death associated with the induction of apoptosis. These findings highlight the role of the class A bHLH proteins as general negative regulators of cell proliferation through a mechanism(s) that involves both enhancement of several cyclin-dependent kinase inhibitor genes expression and promotion of cell death.

Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses and remission of lesions associated with antigen immunotherapy.

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.

Isolation and characterization of four type-1 ribosome-inactivating proteins, with polynucleotide:adenosine glycosidase activity, from leaves of Phytolacca dioica L.

Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29,407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the beta-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues between the two forms. Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had M(r) values of 32,715 +/- 1 (PD-L1), 31,542 +/- 1 (PD-L2), 30,356 +/- 1 (PD-L3) and 29,185 +/- 1 Da (PD-L4). The 1171 kDa difference in M(r), within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin 1 and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.

B-MYB transactivates its own promoter through SP1-binding sites.

B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family.

Retinoblastoma protein tethered to promoter DNA represses TBP-mediated transcription.

The retinoblastoma (RB) tumour suppressor protein negatively regulates cell proliferation by modulating transcription of growth-regulatory genes. Recruitment of Rb to promoters, by association with E2F complex or by fusion with heterologous DNA-binding domains, demonstrated that Rb represses directly transcription. Recent studies also suggest that the RB protein is able to repress gene transcription mediated by the RNA polymerase I and III. Since the TATA-binding protein (TBP) is an important component for transcription mediated by all three RNA polymerases, we have analysed the functional interaction between Rb and TBP in vivo in the context of RNA pol II-driven transcription. We demonstrated that in mammalian cells Rb tethered to promoter represses TBP-mediated activation in vivo, and Rb-mediated repression is reversed in the presence of the inhibition of histone deacetylase activity by trichostatin A (TSA).

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of INF-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.

T-cell responsiveness of American cutaneous leishmaniasis patients to purified Leishmania pifanoi amastigote antigens and Leishmania braziliensis promastigote antigens: immunologic patterns associated with cure.

Patients suffering from American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) or by three proteins (A-2/P-2, P-4, and P-8) derived from Leishmania pifanoi amastigotes were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma), interleukin 2 (IL-2) and interleukin 4 (IL-4) produced were also determined. Results show two different patterns of Lb-induced T cell responses: (a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma, IL-2, and IL-4) during the active disease, (b) similar proportions of responding CD4+ and CD8+ cells and type 1 cytokine production (presence of IFN-gamma and IL-2 and very low IL-4) at the end of therapy (healed lesions). Thus, this last pattern is probably associated with a beneficial T cell response. The A-2/P-2 amastigote cysteine proteinase provided only marginal (s.i. approximately or = 2.5) T cell stimulation in 25% of patients studied; in contrast, the L. pifanoi P-4 and P-8 amastigote antigens induced significant stimulation (s.i. approximately or = 5) in approximately 50% of the patients. In comparison to Lb-stimulated cultures, lower proliferative responses of T lymphocytes to P-4 or P-8 were observed. However, the P-4- or P-8-stimulated cultures had similar percentages of reactive CD4+ and CD8+ cells, as well as type 1 cytokines (presence of IFN-gamma and IL-2, and low levels or absence of IL-4) in the supernatants both before and at the end of therapy. The consistent induction of apparently beneficial T cell responses by the P-4 and P-8 amastigote glycoproteins points to the possibility that these molecules be considered as candidates for future defined vaccines against leishmaniasis.