Baseline serum TSH levels predict the absence of thyroid dysfunction in cancer patients treated with immunotherapy.

PURPOSE: Immunotherapy against immune checkpoints has significantly improved survival both in metastatic and adjuvant setting in several types of cancers. Thyroid dysfunction is the most common endocrine adverse event reported. Patients who are at risk of developing thyroid dysfunction remain to be defined. We aimed to identify predictive factors for the development of thyroid dysfunction during immunotherapy. METHODS: This is a retrospective study including a total of 68 patients who were treated with immune checkpoint inhibitors (ICIs) for metastatic or unresectable advanced cancers. The majority of patients were treated with anti-PD1 drugs in monotherapy or in combination with anti-CTLA4 inhibitors. Thyroid function and anti-thyroid antibodies, before starting immunotherapy and during treatment, were evaluated. Thyroid ultrasound was also performed in a subgroup of patients at the time of enrolment in the study. RESULTS: Eleven out of 68 patients (16.1%) developed immune-related overt thyroid dysfunction. By ROC curve analysis, we found that a serum TSH cut-off of 1.72 mUI/l, at baseline, had a good diagnostic accuracy in identifying patients without overt thyroid dysfunction (NPV = 100%, p = 0.0029). At multivariate analysis, both TSH and positive anti-thyroid antibodies (ATAbs) levels, before ICIs treatment, were independently associated with the development of overt thyroid dysfunction during immunotherapy (p = 0.0001 and p = 0.009, respectively). CONCLUSIONS: Pre-treatment serum TSH and ATAbs levels may help to identify patients at high risk for primary thyroid dysfunction. Our study suggests guidance for an appropriate timely screening and for a tailored management of thyroid dysfunctions in patients treated with ICIs.

Should familial disease be considered as a negative prognostic factor in micropapillary thyroid carcinoma?

PURPOSE: An increased aggressiveness of familial papillary thyroid carcinoma (FPTC) compared with sporadic form has been reported. On the contrary, the biological behavior of familial microPTC (FmPTC) is still debated. To assess if familial diseases should be considered as a negative prognostic factor in mPTC, the clinical presentation and outcome of FmPTC and sporadic mPTC (SmPTC) were compared. METHODS: We retrospectively analyzed 291 mPTC (SmPTC n = 248, FmPTC n = 43) patients followed for a median follow-up of 8.3 years. FmPTC was defined as the presence of PTC in two or more first-degree relatives, after excluding hereditary syndromes associated with PTC. RESULTS: FmPTC patients had more frequently bilateral tumor (32.6% versus 16.5%, p = 0.01) and lymph node metastases at diagnosis (30.2% versus 14.9%, p = 0.02). At the first follow-up, FmPTC patients had a higher rate of structural disease and a lower rate of remission compared to SmPTC (p = 0.01). Also in a multivariate model, using a "CHAID tree-building algorithm", familial disease correlated with a worse clinical presentation and outcome of mPTC patients. Familial disease was associated with a higher rate of intermediate risk patients in non incidental mPTC and with a higher rate of structural incomplete response in mPTC without lymph node metastases (p = 0.01). CONCLUSIONS: Like in macroPTC, the familial form of the diseases has been shown to be a negative prognostic factor also in mPTC, therefore, it should be highly regarded in the management of mPTC patients.

Autoimmune thyroid diseases are more common in patients with prolactinomas: a retrospective case-control study in an Italian cohort.

BACKGROUND: Prolactin may exert immunological effects. Over the years, a higher prevalence of autoimmune thyroid diseases (ATD) has been reported in patients with prolactinomas (PRLs) in areas with sufficient iodine intake. PURPOSE: The aim of our study was to evaluate the prevalence of ATD [Graves' disease (GD) and chronic autoimmune thyroiditis (AIT)] in a retrospective cohort of Italian patients with PRLs compared to a sex-matched control group, represented by subjects with non-functioning pituitary adenoma (NFPA) or empty sella (ES). MATERIALS AND METHODS: We enrolled 149 patients (108 F/41 M) with PRLs (110 micro/39 macro) and 143 subjects (100 F/43 M) with NFPA (n = 96, 56 micro/40 macro) or ES (n = 47), with normal serum prolactin. Neck ultrasound and thyroid function tests (anti-thyroid antibodies, TSH, FT3 and FT4) were performed in all patients. RESULTS: In PRLs, median serum prolactin was significantly higher (98.3 vs. 8.9 ng/ml, p ≤ 0.0001), while age was lower (34 vs. 46 years, p ≤ 0.001) compared to controls. The prevalence of ATD was 13.4% (20/149) in PRLs (1 GD and 19 AIT) compared to 6.3% (9/143) in the controls (p = 0.042). At the multivariate analysis, serum prolactin was the only independent factor predicting ATD. Thyroid volume (12.5 ± 5.9 ml vs. 12.8 ± 10 ml, p = 0.47) and the presence of uni- or multinodular goiter (29.5% vs. 35%, p = 0.35) did not differ between PRLs and control groups. CONCLUSIONS: Our data in an area with mild iodine deficiency confirm a higher prevalence of ATD in patients with prolactinomas.

A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma.

The development of a novel enzyme-linked immunosorbent assay (ELISA) for determining luteinizing hormone (LH) in bovine plasma is described. Anti-bovine LH (bLH) monoclonal antibodies (mAbs) were produced and characterized. One mAb recognizing the bLH ß subunit was used for immunoaffinity purification of substantial amounts of biologically active bLH from pituitary glands. The purified bLH in combination with 2 anti-bLH ß subunit mAbs was used to develop a sandwich ELISA, which satisfied all the criteria required to investigate LH secretory patterns in the bovine species. The ELISA standard curve was linear over the range 0.05 to 2.5 ng/mL, and the assay proved suitable for measuring bLH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 3.41% and 9.40%, and 9.29% and 15.84%, respectively. The analytical specificity of the method was validated in vivo by provocative tests for LH in heifers, using the LH releasing peptide gonadotropin-releasing hormone. In conclusion, the adoption of mAbs for this ELISA for coating the wells and labeling, combined with the easy one-step production of reference bLH, ensures long-term continuity in large-scale measurements of LH in the bovine species.

A collaboration of aquaporins handles water transport in relation to the estrous cycle in the bitch uterus.

Fluid movement through uterine cell membranes is crucial, as it can modulate the tissue imbibition pattern in the different phases of the estrous cycle. To gain insight into the mechanisms underlying steroid-controlled water handling, the presence and distribution of aquaporins (AQPs), integral membrane channel proteins permitting rapid passive water movement, was explored in bitch uterine tissues. Immunohistochemistry and Western immunoblot analysis were used to study the presence of AQP1, AQP2, and AQP5 in the layers of the bitch uterine wall during the different estrous phases. Presence of endothelial nitric oxide-generating enzyme NO synthase (NOS3) was also investigated, as it is known that the vasodilator NOS3 might be involved in the development of uterine edema. The results demonstrated the following: (1) AQP1, AQP2, and AQP5 were present in the uterus of cycling bitches. (2) AQP1 was localized within uterine mesometrial, myometrial, and endometrial blood vessels and in the circular and longitudinal layers of myometrium. AQP1 localization and expression were unaffected by the estrous cycle. (3) The estrogenic milieu was probably at the basis of AQP2 expression in the glandular and luminal epithelium of the endometrium. (4) AQP5 water channels were present in the apical plasma membrane of uterine epithelial cells in coincidence with plasma progesterone increase. (5) NOS3 was localized in the myometrial and epithelial tissues as well as in blood vessels indicating a contribution of this vasoactive peptide to the uterine imbibition processes. Thus, we can hypothesize that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different functional uterine phases.

Oxacilin-resistant Coagulase-negative staphylococci (CoNS) bacteremia in a general hospital at São Paulo city, Brasil / Bacteremias por Staphylococcus coagulase negativos oxacilina resistentes em um hospital na cidade de São Paulo, Brasil

In the last decades, coagulase-negative staphylococci (CoNS), especially Staphylococcus epidermidis have become an important cause of bloodstream infections. In addition, rates of methicillin-resistance among CoNS have increased substantially, leading to the use of glicopeptides for therapy. The objective of this study was to evaluate eleven consecutives clinically relevant cases of oxacillin-resistant CoNS bacteremia in a general hospital localized in São Paulo city, Brazil. Five different species were identified by different phenotypic methods, including S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) and S. cohnii subsp urealyticus (1). A variety of Pulsed Field Gel Electrophoresis profiles was observed by macrorestriction DNA analysis in S. epidermidis isolates, but two of three S. haemolyticus isolates presented the same profile. These data indicated the heterogeneity of the CoNS isolates, suggesting that horizontal dissemination of these microorganisms in the investigated hospital was not frequent. One S. epidermidis and one S. haemolyticus isolates were resistant to teicoplanin and susceptible to vancomycin. The selective pressure due to the use of teicoplanin in this hospital is relevant.

Staphylococcus coagulase negativos (SCoN), especialmente Staphylococcus epidermidis tem se tornado causa importante de infecções da corrente circulatória nas últimas décadas. Além disso, percentuais de resistência a meticilina entre os SCoN têm aumentado significativamente, levando ao uso de glicopeptídeos nestes pacientes. O objetivo deste estudo foi avaliar onze casos consecutivos de bacteremia clinicamente relevantes por SCoN oxacilina resistentes em um hospital localizado na cidade de São Paulo, Brasil. Cinco diferentes espécies foram identificadas por diferentes métodos fenotípicos, incluindo S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) e S. cohnii subsp urealyticus (1). Diferentes perfis eletroforéticos obtidos pela técnica de "Pulsed Field Gel Electrophoresis" foram observados na análise da macrorestrição do DNA nos isolados de S. epidermidis, mas dois dos três isolados de S. haemolyticus apresentaram o mesmo perfil. Esses dados indicam uma heterogeneidade nos isolados SCoN, sugerindo que a disseminação horizontal no hospital investigado não é freqüente. Um isolado de S. epidermidis e um de S. haemolyticus foram resistentes à teicoplanina e sensíveis à vancomicina. Observa-se a relevância da pressão seletiva pelo uso de teicoplanina nos pacientes deste hospital.

Oxacilin-resistant Coagulase-negative staphylococci (CoNS) bacteremia in a general hospital at São Paulo city, Brasil.

In the last decades, coagulase-negative staphylococci (CoNS), especially Staphylococcus epidermidis have become an important cause of bloodstream infections. In addition, rates of methicillin-resistance among CoNS have increased substantially, leading to the use of glicopeptides for therapy. The objective of this study was to evaluate eleven consecutives clinically relevant cases of oxacillin-resistant CoNS bacteremia in a general hospital localized in São Paulo city, Brazil. Five different species were identified by different phenotypic methods, including S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) and S. cohnii subsp urealyticus (1). A variety of Pulsed Field Gel Electrophoresis profiles was observed by macrorestriction DNA analysis in S. epidermidis isolates, but two of three S. haemolyticus isolates presented the same profile. These data indicated the heterogeneity of the CoNS isolates, suggesting that horizontal dissemination of these microorganisms in the investigated hospital was not frequent. One S. epidermidis and one S. haemolyticus isolates were resistant to teicoplanin and susceptible to vancomycin. The selective pressure due to the use of teicoplanin in this hospital is relevant.

Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence.

In the last few years, several works suggest that Growth Hormone (GH) is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH. We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs) and the concentration of GH in the oocytes and in the follicular fluids (FF) from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi) show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo). At the same time we measured Estrogen (E2) and Progesterone (P4) concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA). We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrine-paracrine manner. Moreover, E2, and P4 levels in FF suggest that, in our model, atresia processes are also involved in oocyte developmental capability and that the highest level of GH may represent a local reaction to these phenomena.

Heterophile antibody interference in a solid phase sandwich immunoassay for detection of equine growth hormone in plasma.

Heterophile antibodies (HAs) present in serum recognize animal immunoglobulins and are one of the most unpredictable causes of false results in immunoassays. However, no study has yet reported their interference on the diagnostic reliability of immunochemical analyses on horse plasma. Recently, we developed a sandwich ELISA for detection of equine growth hormone (eGH) in plasma. In a pilot study to measure basal eGH levels (blood samples were drawn from 13 horses every 10 min for 1h), we noted one horse with abnormally high eGH (>100 ng/mL). We demonstrate here that this plasma eGH level was falsely elevated due to interference from HAs. The interfering antibodies were polyspecific immunoglobulins, with fairly broad species-specificity, which affected the eGH immunoassay by bridging the mouse IgG capture antibody and the rabbit IgG conjugate. This produced artificial sandwiches which led to overestimation of the eGH plasma concentration. Spiking horse plasma with pure mouse and rabbit immunoglobulins or whole plasma of several species significantly reduced but did not totally eliminate the HAs interference. Immunoglobulins and whole plasma differed in their ability to block the interference, suggesting that HAs may recognize other proteins beside immunoglobulins in animal sera. To investigate whether HAs have any implications in equine clinical practice, we decided to seek information on the incidence of HAs interference in normal animals. We collected single plasma samples from another 114 horses and we found that 5 of these had plasma HAs. Therefore, in total 6 out of the 127 horses examined (4.7%) had plasma HAs generating falsely elevated eGH measures. In conclusion, this study provides the first evidence of HAs in horse plasma interfering with an immunoassay and indicates that veterinary surgeons and diagnostic laboratory staff should be aware of this potential for interference in tests on horse plasma using monoclonal or polyclonal antibody reagents.

Effects of disease progression and L-dopa therapy on the control of reaching-grasping in Parkinson's disease.

The present study aimed to determine whether the bradykinesia of Parkinson's disease (PD) patients during the execution of reaching-grasping movements (i) is related to an impaired implementation of movement parameters and (ii) selectively involves the control of reach and/or grasp movements. We compared the kinematics of reaching to grasp of differently sized objects placed at different positions, among PD patients in the early stage of disease (ESPD), in the advanced stage of disease (ASPD) without L-dopa medication (off-state), and in healthy controls. In addition, we analysed the effects of L-dopa replacement therapy by comparing the kinematics of the patients in the advanced stage of disease after L-dopa administration with those of the other groups. Bradykinesia increased with disease progression, but only in the initial phases of the reach and grasp components. However at both stages of the disease, the kinematics of reaching and grasping responded to extrinsic and intrinsic object properties just as in controls. L-dopa administration improved the performance of PD patients, though this was more evident for the reach than for the grasp. We suggest that the basal ganglia (BG) are involved in implementing kinematic parameters, but neither (or only marginally) in the initial movement parameterization itself, nor in the on-line control of movement. Specifically, the BG dysfunction in PD induces a slowed implementation of movement parameters. The lack of effect of L-dopa administration on grasp kinematics may be because the motor control of distal effectors is less represented in the motor circuitry formed by the supplementary motor area (SMA), thalamus and BG.

Identification of commercial fish species belonging to the orders pleuronectiformes and gadiformes: library of isoelectric focusing patterns.

Isoelectric focusing in polyacrylamide gel was used to establish an identification archive of fish species belonging to the orders Pleuronectiformes, or flat fish, and Gadiformes, or gadoid fish. The 2 orders include species of different commercial value and interest that are frequently requested in European fish markets, but are susceptible to substitution either because they are morphologically similar or because they arrive on the markets already filleted or sliced. The sarcoplasmic protein profiles are species-specific and reproducible. The use of densitometry and image analysis coupled with a simple computer program overcomes the subjective evaluation of the patterns, making it possible to identify species correctly.

Amino acid modifications in canine, equine and porcine pituitary growth hormones, identified by peptide-mass mapping.

Modified amino acid residues in porcine, canine and equine growth hormones purified from pituitary glands were characterised by tryptic mapping and high-performance liquid chromatography with on-line coupled electrospray ionisation mass spectrometry (HPLC-ESI-MS) detection. Hormones from all three species showed the same changes. Conversion of Asp128 to iso-Asp128 was a component of native hormones, while deamidation of Asn12 and Asn98 to Asp and iso-Asp, oxidation of Met4, and cyclisation to the pyroglutamyl derivative of Gln139, probably occurred in vitro, during isolation, storage or hydrolysis. Porcine and canine hormones had indistinguishable protein fingerprints, confirming the assumption, based on their cDNA sequences, that their mature primary structures are identical.

Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems.

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.

Binding and signalling properties of a growth hormone enhancing monoclonal antibody.

We have used a sequential, qualitative biosensor based assay to demonstrate that OA15, a monoclonal antibody which enhances in vivo the activity of bovine growth hormone (bGH) does not disrupt the interaction between bGH and its cognate receptor (as represented by recombinant bovine GH binding protein -rbGHBP). We have confirmed this using a classical cell-based radio-receptor assay with the GH-responsive mouse pre-adipocyte cell line 3T3-F442A. The fact that OA 15 binding to bGH still allows hormone to interact with its receptor, allows us to test the hypothesis that there is any amplification of signalling events following hormone-MAb treatment of 3T3-F442A cells. We have used as a reporter of GH activity the rapid stimulation of JAK-2 tyrosine phosphorylation which is a critical first step in GH signalling events. We demonstrate that binding of rbGH by OA15 attenuates hormone stimulation of JAK-2 tyrosine phosphorylation. We conclude that although OA15 does not disrupt GH-GH receptor (GHR) interactions it does interfere with subsequent GH activity at the molecular and cellular level. We further speculate therefore that the biological enhancing activity of this antibody is most likely due to an in vivo effect as presentation of antibody-hormone complexes to a GH-target cell inhibits hormone activity.