Could different aqueous humor and plasma cytokine profiles help differentiate between ocular sarcoidosis and ocular tuberculosis?

OBJECTIVE AND DESIGN: A cross-sectional single-center study was conducted to assess cytokine levels in aqueous humor (AH) and plasma of three different uveitis entities: definite ocular sarcoidosis (OS), definite OS associated with QuantiFERON®-TB Gold test positivity (Q + OS) and presumed tubercular uveitis (TBU). SUBJECTS: Thirty-two patients (15 OS, 5 Q + OS, 12 TBU) were included. METHODS: Quantification of selected cytokines was performed on blood and AH samples collected before starting any treatment. Statistical analysis was conducted using the Kruskal-Wallis test, the Mann-Whitney or Fisher test and the Principal Component Analysis (PCA). RESULTS: IL-6, IL-8 and IP-10 levels were higher in AH samples than in peripheral blood. In AH samples, BLC, IL-8 and IP-10 were significantly higher in definite OS than in presumptive TBU. There were no statistically significant differences in terms of cytokine levels between Q + OS and presumptive TBU. PCA showed a similar cytokine pattern in the latter two groups (IFNγ, IL-15, IL-2, IP-10, MIG), while the prevalent expression of BLC, IL-10 and MIP-3 α was seen in definite OS. CONCLUSIONS: The different AH and plasma cytokine profiles observed in OS compared to Q + OS and TBU may help to differentiate OS from TBU in overlapping clinical phenotypes of granulomatous uveitis (Q + OS).

Vogt-Koyanagi-Harada patients show higher frequencies of circulating NKG2Dpos NK and NK T cells.

Vogt-Koyanagi-Harada (VKH) is an autoimmune disease characterized by inflammation in tissues that contain melanocytes. We aimed to increase the knowledge regarding immunological pathways deregulated in VKH disease. We compared the percentages of circulating natural killer (NK), NK T and T cells expressing the activatory markers: CD16, CD69, NK group 2D (NKG2D), natural cytotoxicity triggering receptor 3 (Nkp30), natural cytotoxicity triggering receptor 1 (Nkp46) and the inhibitory marker: NK group 2 member A (NKG2A) in 10 active VKH patients, 20 control subjects (CTR) and seven patients with Behçet disease (BD) by flow cytometry. Cytotoxic potential of NK cells was determined through the degranulation marker CD107a expression after contact with K562 cells by flow cytometry. Moreover, plasmatic levels of 27 cytokines were determined with a multiplex bead-based assay. VKH patients showed higher percentages of NKG2Dpos NK and NK T cells versus CTR. The cytotoxic potential of NK cells induced by K562 cells was comparable between VKH patients and CTR. Finally, higher concentrations of interleukin (IL)-4, IL-5, IL-7, IL-17 and platelet-derived growth factor-subunits B (PDGF-bb) were detected in plasma of VKH patients versus CTR. The immune profile of VKH patients was similar to that of BD patients.

Aqueous tap and rapid diagnosis of cytomegalovirus anterior uveitis: the Reggio Emilia experience.

PURPOSE: The diagnosis of cytomegalovirus (CMV) anterior uveitis in immunocompetent patients requires confirmation by polymerase chain reaction (PCR) analysis and/or intraocular antibody index (AI) assay. In this study, we analyzed the different contributions of PCR and AI to CMV diagnosis by performing one single aqueous tap. METHODS: A retrospective chart review was conducted of HIV-negative patients attending the Ocular Immunology Unit of Azienda Unità Sanitaria Locale - IRCCS, Reggio Emilia, Italy, from March 2015 to April 2018 with a diagnosis of hypertensive anterior granulomatous uveitis compatible with suspected CMV etiology. Diagnosis was confirmed by real-time PCR (RT-PCR) and intraocular antibody production against CMV on aqueous humor samples. Clinical features were compared to antibody titer and diagnostic delay. RESULTS: Twenty-three patients with suspected CMV uveitis (13 males, 10 females, mean age 48 ± 16 years) were included in the analysis. AI was positive in 20/23 (87%) samples, and PCR tested positive in 9/23 (39%). By combining both tests, the sensitivity was 100%. Median diagnostic delay was 29 months (IQR 9-107). Diagnostic delay and antibody titer were significantly associated with glaucoma (r = 0.714, p < 0.0001; r = 0.476, p = 0.02, respectively). CONCLUSIONS: Our data suggest that to improve the diagnostic accuracy of CMV anterior uveitis, PCR and AI are both useful and complimentary. In our series, AI was the most sensitive diagnostic tool. One single aqueous tap is sufficient to achieve 100% sensitivity in CMV diagnosis. Early diagnosis is necessary to prevent the development of glaucoma.

Role of immunoglobulin G fragment C receptor polymorphism-mediated antibody-dependant cellular cytotoxicity in colorectal cancer treated with cetuximab therapy.

Antibody-dependent cellular cytotoxicity (ADCC), which is activated by effector cells via immunoglobulin G (IgG) fragment C receptors (FcRs), was proposed as a mechanism of cetuximab efficacy. Peripheral blood mononuclear cells (PBMCs) from 23 healthy donors and 13 patients with metastatic colorectal cancer (mCRC) treated with cetuximab were tested for FcγR polymorphisms and cetuximab-mediated ADCC. ADCC was measured by chromium-51 release on a epidermal growth factor receptor (EGFR)-positive human colon cancer cell line. Overall, 86 mCRC patients were genotyped for study purposes. PBMCs harbouring the FcγRIIIa 158 V/V genotype had a significantly higher cetuximab-mediated ADCC. No correlation was found between FcγR polymorphisms and response rate or time to progression after cetuximab-based therapy. Despite the in vitro analysis showing that the FcγRIIIa 158 V/V genotype is associated with higher ADCC, clinical data do not support a predictive role of FcγRIIIa polymorphisms in mCRC treated with cetuximab.

Reggio Emilia (Northern Italy) Interdisciplinary Uveitis Clinic: What We Have Learned in the Last 20 Years.

PURPOSE: To analyze the referral patterns and the clinical and therapeutic features of patients diagnosed with uveitis in an Italian tertiary referral center to provide a comparison with previously published series from the same center. METHODS: Retrospective retrieval of data on all new referrals to the Ocular Immunology Unit in Reggio Emilia (Italy) between November 2015 and April 2022 and comparison with previously published series from the same center. RESULTS: Among the 1557 patients, the male-to-female ratio was 1:1.27. Anterior uveitis was the most common diagnosis (53.7%), followed by posterior (21.6%), pan- (18.5%), and intermediate (6.2%) uveitis. The most identifiable specific diagnoses were anterior herpetic uveitis (18.4%), Fuchs uveitis (12.8%), and tuberculosis (6.1%). Infectious etiologies were the most frequent (34.1%) and were more diffuse among non-Caucasian patients (p < 0.001), followed by systemic disease-associated uveitis (26.5%), and ocular-specific conditions (20%). Idiopathic uveitis accounted for 19.4% of cases. Fuchs uveitis presented the longest median diagnostic delay (21 months). Immunosuppressants were administered to 25.2% of patients. Antimetabolites, calcineurin inhibitors, and biologicals were prescribed to 18.4%, 3%, and 11.4% of cases, respectively. Compared to our previous reports, we observed a significant increase in foreign-born patients and in infectious uveitis, a decrease in idiopathic conditions, and an increasing use of non-biological and biological steroid-sparing drugs. CONCLUSIONS: The patterns of uveitis in Italy have been changing over the last 20 years, very likely due to migration flows. Diagnostic improvements and a more widespread interdisciplinary approach could reduce the incidence of idiopathic uveitis as well as diagnostic delay.

Diversity and dynamics of ammonia-oxidizing bacterial communities in a sponge-based trickling filter treating effluent from a UASB reactor.

Changes in ammonia-oxidizing bacterial (AOB) population dynamics were examined in a new sponge-based trickling filter (TF) post-upflow anaerobic sludge blanket (UASB) reactor by denaturating gradient gel electrophoresis (DGGE), and these changes were linked to relevant components influencing nitrification (chemical oxygen demand (COD), nitrogen (N)). The sponge-based packing media caused strong concentration gradients along the TF, providing an ecological selection of AOB within the system. The organic loading rate (OLR) affected the population dynamics, and under higher OLR or low ammonium-nitrogen (NH4(+)-N) concentrations some AOB bands disappeared, but maintaining the overall community function for NH4(+)-N removal. The dominant bands present in the upper portions of the TF were closely related to Nitrosomonas europaea and distantly affiliated to Nitrosomonas eutropha, and thus were adapted to higher NH4(+)-N and organic matter concentrations. In the lower portions of the TF, the dominant bands were related to Nitrosomonas oligotropha, commonly found in environments with low levels of NH4(+)-N. From a technology point of view, changes in AOB structure at OLR around 0.40-0.60 kgCOD m(-3) d(-1) did not affect TF performance for NH4(+)-N removal, but AOB diversity may have been correlated with the noticeable stability of the sponge-based TF for NH4(+)-N removal at low OLR. This study is relevant because molecular biology was used to observe important features of a bioreactor, considering realistic operational conditions applied to UASB/sponge-based TF systems.

Early kinetics of innate and adaptive immune responses during hepatitis B virus infection.

BACKGROUND AND AIMS: Innate immunity appears to be silent in acutely hepatitis B virus (HBV)-infected chimpanzees, as shown by microarray analysis of intrahepatic gene expression. Whether this observation also applies to HBV pathogenesis in man remains undefined. The aim of this study was thus to characterise natural killer (NK) and CD56(+) natural T (NT) cell responses early after human HBV infection and their relationship to the induction of adaptive immunity. METHODS: Two HBV-seronegative blood donors who became hepatitis B surface antigen (HBsAg) and HBV DNA positive but had persistently normal alanine aminotransferase (ALT) were followed from a very early stage of HBV infection. The phenotype (CD69 and NKG2D) and function (cytotoxicity and interferon gamma (IFN gamma) production) of NK and NT cells were analysed. CD4- and CD8-mediated responses were studied in parallel with overlapping peptides covering the entire HBV sequence by ex vivo intracellular cytokine staining (ICS) for IFN gamma, interleukin 2 (IL2), IL4 and IL10, and by ex vivo Elispot for IFN gamma. Healthy subjects, and patients with chronic and acute HBV infection were studied for comparison. RESULTS: An early induction of both innate and adaptive responses was observed. NK and NT cells showed faster kinetics than HBV-specific T cells with an earlier peak of activity, while CD4(+) and CD8(+) cell responses were mounted with a similar profile, with higher frequencies of IFN gamma-producing CD8(+) cells at the peak of the response. CONCLUSIONS: The innate immune system is able to sense HBV infection, as shown by the early development of NK and NT cell responses, which probably contribute to contain the HBV infection and to allow timely induction of adaptive responses.

Is there a role for immunotherapy in hepatocellular carcinoma?

Incidence of hepatocellular carcinoma has been rising in the last two decades because of the wide exposure to hepatitis C virus during 1960s and 1970s. Improvement in treatment has been achieved by local ablative therapies, however because of early recurrence and lack of effective chemotherapies, alternative treatments based on stimulation of the anti-tumour immune response could represent new strategies to control hepatocellular carcinoma spread and recurrence. Proof of principle of an effective immunotherapy has been achieved for other solid tumours such as melanoma and several results could be transferred to the immunotherapy of hepatocellular carcinoma. Specific tumour antigens have been identified in hepatocellular carcinoma, such as cancer testis antigens expressed in a large part of hepatocellular carcinomas and alpha-fetoprotein that has been already employed in clinical trials demonstrating immunogenicity without however significant clinical efficacy. Better results have been achieved by non-antigen-specific immunotherapies that demonstrated improvement in recurrence and recurrence-free survival in patients undergoing surgical resection for hepatocellular carcinoma. Passive immunotherapy and targeted therapies blocking tumour cell receptors or enzymatic pathways are already in the clinic for other malignancies and the near future will see these new treatments applied to hepatocellular carcinoma patients along with the development of efficacious active immunotherapies aimed at reducing disease recurrence and improving survival.

Glutamine deprivation-mediated cell shrinkage induces ligand-independent CD95 receptor signaling and apoptosis.

Cell shrinkage and loss of cell viability by apoptosis have been examined in cultured CD95(Fas/Apo-1)-expressing leukemia-derived CEM and HL-60 cells subjected to acute deprivation of glutamine, a major compatible osmolyte engaged in cell volume control. Glutamine deprivation-mediated cell shrinkage promoted a ligand-independent activation of the CD95-mediated apoptotic pathway. Cell transfection with plasmids expressing FADD-DN or v-Flip viral proteins pointed to a functional clustering of CD95 receptors at the cell surface with activation of the 'extrinsic pathway' caspase cascade. Accordingly, cell shrinkage did not induce apoptosis in CD95 receptor-negative lymphoma L1210 cells. Replacement of glutamine with surrogate compatible osmolytes counteracted cell volume decrement and protected the CD95-expressing cells from apoptosis. A glutamine deprivation-dependent cell shrinkage with activation of the CD95-mediated pathway was also observed when asparaginase was added to the medium. Asparagine depletion had no role in this process. The cell-size shrinkage-dependent apoptosis induced by glutamine restriction in CD95-expressing leukemic cells may therefore be of clinical relevance in amidohydrolase enzyme therapies.

Valuation and modelling of helminth eggs removal in baffled and unbaffled ponds treating anaerobic effluent.

The paper evaluates and models helminth eggs removal in a combined UASB (Upflow Anaerobic Sludge Blanket) reactor-maturation pond system (one baffled and one unbaffled pond in parallel). The system is comprised of demonstration-scale units, treating actual domestic sewage from Itabira city, Brazil. The paper addresses the following points: (i) removal of helminth eggs from the wastewater in the system; (ii) comparison of the observed removal efficiency with the predictions using the Ayres et al. model; (iii) accumulation of helminth eggs in the sludge; (iv) viability of eggs in the sludge; (v) distribution of helminth species in the sludge. The removal efficiency of helminth eggs from the wastewater in both ponds was 100% for most of the time (0 counts in the final effluent). The predictions of helminth eggs removal according to the Ayres et al. model can be considered reasonable. After one year of operation, 88% of the eggs in the sludge remained viable. Helminth eggs counts in the sludge tended to decrease along the baffled pond length (from first to last compartment). The prevailing helminth species found in the sludge from both ponds was Ascaris lumbricoides.

Post-treatment of anaerobic effluents in an overland flow system.

This research aimed at the investigation of an overland flow system applied to the post-treatment of anaerobic effluents. The system treated domestic sewage in Itabira City (Brazil), being composed by an anaerobic reactor and an overland flow system, the latter working as a post-treatment unit. A portion of the reactor's effluent was directed to a group of three overland flow slopes (demonstration scale), that were operated with different application rates. During Phase 1 of the research the overland flow system was fed under a permanent hydraulic regime (constant flows), having as inflow the effluent from an UASB reactor (full-scale, volume of 477 m3). During Phase 2, the overland system was fed under a hydraulic transient pattern (variable flows with hourly variations), having as inflow the effluent from a partitioned UASB reactor (demonstration-scale, volume of 9 m3). In general, the performance of the overland flow system as a polishing step was very good, mainly because of the low solids and organic matter concentration in the final effluent (average values of BOD from 48 to 62 mg/L; COD from 98 to 119 mg/L and SS from 17 to 57 mg/L). Regarding nutrients and coliforms, the system also reached satisfactory efficiency levels. Based on the experience obtained with this study, it is suggested that overland flow systems, working as post-treatment step of UASB reactors, can work with application rates in the range of 0.4 to 0.5 m3/m.h, which are higher than those normally applied.

Inactivation of E. coli and helminth eggs in aerobic and anaerobic effluents using UV radiation.

This paper evaluates the performance of a simplified bench-scale UV-photoreactor used to inactivate Escherchia coli and eggs of Ascaris lumbricoides. The photoreactor consisted of a tubular unit constructed with PVC tube, 100 mm diameter and 45 cm total height, with a low-pressure mercury lamp adapted in the centre of the tube. The reactor was tested to disinfect the effluent from a trickling filter and from an UASB reactor, both fed with domestic sewage. The results showed an excellent performance of the photoreactor, with very high E. coli inactivation efficiencies being observed for the aerobic effluent (in the range of 4 to 5 log-units, for doses varying from 50.7 to 13.6 mW x s x cm(-2)) and also for the effluent from the UASB reactor (usually above 4 log-units, for doses of 20.3 and 13.6 mW x s x cm(-2)). In relation to the inactivation of helminth eggs, it was observed that UV radiation significantly affected the development of eggs of Ascaris lumbricoides, with the better results being obtained for radiation times of 40 and 60 seconds (doses of 13.6 and 20.3 mW x s x cm(-2), respectively), when approximately 65% of the eggs remained in the stage of single cell and only 9 to 10% were able to fully develop to the stage of motile larva.

Coliform and helminth eggs removal in a combined UASB reactor-baffled pond system in Brazil: performance evaluation and mathematical modelling.

The paper presents the monitoring results of a pilot UASB reactor followed by a baffled polishing pond treating domestic sewage in Brazil. Longitudinal profiles of E coli and helminth eggs along the baffled pond have been undertaken. The experimental results have been compared with von Sperling's model for coliform removal and Ayres' model for helminth eggs removal, and the fitting was considered satisfactory in both cases. The distribution of the helminth species along the system is also presented.

L-Asparaginase and inhibitors of glutamine synthetase disclose glutamine addiction of ß-catenin-mutated human hepatocellular carcinoma cells.

Selected oncogenic mutations support unregulated growth enhancing glutamine availability but increasing the dependence of tumor cells on the amino acid. Data from literature indicate that a subset of HepatoCellular Carcinomas (HCC) is characterized by mutations of ß-catenin and overexpression of Glutamine Synthetase (GS). To assess if this phenotype may constitute an example of glutamine addiction, we treated four human HCC lines with the enzyme L-Asparaginase (ASNase), a glutaminolytic drug. ASNase had a significant antiproliferative effect only in the ß-catenin mutated HepG2 cells, which were partially rescued by the anaplerotic intermediates pyruvate and α-ketoglutarate. The enzyme severely depleted cell glutamine, caused eIF2α phosphorylation, inhibited mTOR activity, and increased autophagy in both HepG2 and in the ß-catenin wild type cell line Huh-7. When used with ASNase, the GS inhibitor methionine sulfoximine (MSO) emptied cell glutamine pool, arresting proliferation in ASNase-insensitive Huh-7 cells and activating caspase-3 and apoptosis in HepG2 cells. Compared with Huh-7 cells, HepG2 cells accumulated much higher levels of glutamine and MSO, due to the higher expression and activity of SNAT2, a concentrative transporter for neutral amino acids, but were much more sensitive to glutamine withdrawal from the medium. In the presence of ASNase, MSO caused a paradoxical maintenance of rapamycin-sensitive mTOR activity in both HepG2 and Huh-7 cells. ß-catenin silencing lowered ASNase sensitivity of HepG2 cells and of Huh-6 cells, another ß-catenin-mutated cell line, which also exhibited high sensitivity to ASNase. Thus, ß-catenin mutated HCC cells are more sensitive to glutamine depletion and accumulate higher levels of GS inhibitors. These results indicate that glutamine deprivation may constitute a targeted therapy for ß-catenin-mutated HCC cells addicted to the amino acid.

Sistemas multiparticulados: minicomprimidos / Multiparticulate systems: minitablets

Os sistemas multiparticulados contêm o fármaco subdividido em unidades funcionais, que podem ser pellets, grânulos ou minicomprimidos. Oferecem vantagens tecnológicas e biofarmacotécnicas quando comparados aos sistemas monolíticos, apresentando ainda benefícios terapêuticos para os pacientes. Por isso, têm se destacado cada vez mais dentre as novas formas farmacêuticas e sistemas de liberação de fármacos. Os minicomprimidos possuem diâmetro igual ou inferior a 2 - 3 mm e são obtidos através do processo de compressão, utilizando máquinas de comprimir convencionais adaptadas com punções múltiplos. Quanto ao aspecto tecnológico, oferecem vantagens em relação aos pellets e grânulos. O objetivo desta revisão é, portanto, abordar os principais aspectos tecnológicos envolvidos na sua obtenção, destacando suas vantagens e aplicações.

In multiparticulate systems, the drug is subdivided into functional units, which may be in the form of pellets, granules or minitablets. They have technological and biopharmacotechnical advantages over monolithic systems and also offer therapeutic benefits to patients. For these reasons, they have gained prominence among new pharmaceutical dosage forms and drug delivery systems. Minitablets are between 2 and 3 mm in diameter or smaller and are manufactured by means of the direct compression process, with a conventional tablet press adapted with multi-tip punches. In technological terms, they have advantages over both pellets and granules. The objective of this review is, therefore, to discuss the main technological features of minitablet production, highlighting their advantages and applications.

[Dyslalia and orofacial dysmorphism]. / Dislalie e dismorfismi oro-facciali.

In the light of reported data and personal experience, relations between malocclusions and dyslalias are examined. Although the coexistence of malocclusions and dyslalia is a frequent clinical observation, correlations between the two conditions do not appear to be closely linked by any clearly identifiable cause-effect relationship. Considering the high number of aetiological factors involved, stress is laid on the need for a multidisciplinary approach to cases that present contemporaneously malocclusions and dyslalias in the interests of correct diagnosis and rational therapeutic orientation.