Cytoreductive treatment in patients with CALR-mutated essential thrombocythaemia: a study comparing indications and efficacy among genotypes from the Spanish Registry of Essential Thrombocythaemia.

The present study assessed the criteria for initiating cytoreduction and response to conventional therapies in 1446 patients with essential thrombocythemia (ET), 267 (17%) of which were CALR-mutated. In low risk patients, time from diagnosis to cytoreduction was shorter in CALR-positive than in the other genotypes (2·8, 3·2, 7·4 and 12·5 years for CALR, MPL, JAK2V617F and TN, respectively, P < 0·0001). A total of 1104 (76%) patients received cytoreductive treatment with hydroxycarbamide (HC) (n = 977), anagrelide (n = 113), or others (n = 14). The estimated cumulative rates of complete haematological response (CR) at 12 months were 40 % and 67% in CALR and JAK2V617F genotypes, respectively. Median time to CR was 192 days for JAK2V617F, 343 for TN, 433 for MPL, and 705 for CALR genotypes (P < 0·0001). Duration of CR was shorter in CALR-mutated ET than in the remaining patients (P = 0·003). In CALR-positive patients, HC and anagrelide had similar efficacy in terms of response rates and duration. CALR-mutated patients developed resistance/intolerance to HC more frequently (5%, 23%, 27% and 15% for JAK2V617F, CALR, MPL and TN, respectively; P < 0·0001). In conclusion, conventional cytoreductive agents are less effective in CALR-mutated ET, highlighting the need for new treatment modalities and redefinition of haematologic targets for patients with this genotype.

Identification of a beta 2 (CD18) molecule in a teleost species, Ictalurus punctatus Rafinesque.

Beta 2, in combination with the alpha subunit, is responsible for tight adhesion of leukocytes, especially neutrophils and macrophages, in areas of inflammation. Although identified in mammalian and avian species; the beta 2 or CD18 molecule has yet to be identified in fish. The present investigation has identified a full-length channel catfish, Ictalurus punctatus, cDNA beta 2 molecule composed of 2.8 kb and a deduced amino acid sequence of 772 amino acids. The catfish molecule has an amino acid homology ranging from 54 to 63% with mouse, bovine, rabbit, human and chicken. The channel catfish molecule retains several characteristics of mammalian beta 2 molecules, such as cysteine-rich repeat regions, N-linked glycosylation sites, and several proposed signal sequences. Expression of the beta 2 molecule on the catfish neutrophil cytoplasmic membranes is increased upon phorbol dibutyrate stimulation of the cells. Based on Western blotting and the immunoprecipitation test, the channel catfish beta 2 molecule has a molecular mass of approximately 95 kD, essentially the same as that for mammalian species. However, two additional molecules, perhaps alpha chains, of unexpected molecular mass appear to co-precipitate in the SPIT with the 95 kD CD18 molecule. These results confirm the existence and expression of a beta 2 gene in channel catfish, a species phylogenetically distant from mammalian species.

Location of superoxide production sites in turbot neutrophils and gilthead seabream acidophilic granulocytes during phagocytosis of glucan particles.

Here we show, by spectrophotometry and enzyme cytochemical methods, that turbot neutrophils and gilthead seabream acidophils responded in a similar way when incubated with PMA or with particulate glucans. Cells stimulated with PMA released high amounts of superoxide both intra- and extracellularly. However, O2- was mainly released intracellularly when cells were incubated with particulate glucans. Small glucan particles were quickly phagocytosed and O2- was initially produced in intracellular vesicles and tubular structures that later fused with the phagosome or with the cell membrane. Large glucan filaments that were not phagocytosed also induced cell stimulation and O2- was also produced in intracellular vesicles which then appeared to fuse with the cell membrane. We conclude that, in stimulated turbot neutrophils and gilthead seabream acidophils, superoxide production is carried out initially in intracellular compartments that are very similar to those described in mammalian neutrophils.

Identification of a channel catfish, Ictalurus punctatus (Rafinesque), leukocyte-specific leucine zipper protein.

Five clones isolated from a channel catfish cDNA library were each reactive with monoclonal antibodies (mAbs) C3-1 and 51A only. The size of the cDNA inserts from C3-1 and 51A positive clones was 2.5 Kb and identical based on sequence analysis. Monoclonal antibodies C3-1 and 51A specifically reacted with the expressed product of the 2.5 Kb cDNA clone. The complete DNA sequence indicated that the 2.5 Kb cDNA encoded an approximately 50 Kd protein molecule consisting of 445 amino acids. Sequence analysis showed that this putative protein was a potential leucine-zipper DNA binding protein. Comparison of the deduced amino acid sequence demonstrated homology (14.6 to 19.5%) throughout the sequence of the catfish protein with a group of cytoplasmic-leucine zipper containing proteins of humans; paraneoplastic cellebellar degeneration related (cdr) antigen 2 and 3 with 39.8 to 56.3% homology in the leucine-zipper motif (amino acids 52 through 175 in the catfish protein). This protein was detected in nuclear extracts. cytoplasmic membrane preparations and cytosolic extracts of neutrophils and lymphocytes when reacted with mAbs C3-1 and 51A in an ELISA. However, the intensity of the reactions was dependent upon the cell type and cellular component. The putative cdr protein was not detected with any appreciable intensity in preparations from other cell types. This finding strongly suggests that this protein is expressed in a leukocyte-specific manner and is unique among the cdr group in that it is being expressed in a site that is not immune privileged.

Adherence and invasive capacities of the fish pathogen Pasteurella piscicida.

Pasteurella piscicida strains were weakly or moderately adherent to cell lines, the levels of attachment being variable depending on the cells employed. All the isolates exhibited the highest binding capacity to CHSE-214 cells. Adhesive capacities were affected by heat and sugars but not by proteinase K or by treatment with antisera raised against the lipopolysaccharides of P. piscicida, implicating components of glycoprotein(s) as ligands in the adhesion process. The isolates showed a great binding capacity to intestines from the marine fish hosts gilthead sea bream, sea bass and turbot, with values ranging from 10(4) to 10(5) bacteria/g. Although the P. piscicida strains showed a weak invasiveness in the poikilothermic cell lines employed as in vitro model, the bacteria remained viable inside the infected cells at least for 2 days. The invasion process was inhibited by cytochalasin D indicating the active participation of the host cytoskeleton in the internalization of P. piscicida.

Molecular characterization and leukocyte distribution of a teleost beta1 integrin molecule.

The beta1 integrin, in combination with the alpha subunit, is responsible for migration of leukocytes into areas of inflammation. Although identified in mammalian species; the beta1 or CD29 molecule has yet to be identified in fish. The present investigation has identified a full-length channel catfish, Ictalurus punctatus, cDNA beta1 molecule composed of 2786 bases and a deduced amino acid sequence of 797 amino acids. The catfish molecule has an amino acid identity ranging from 71.87 to 74.12% with bovine, feline, human, and Xenopus. The channel catfish molecule retains several characteristics of mammalian beta1 molecules, such as four cysteine-rich repeat regions, and eight potential N-linked glycosylation sites. Based on Western blotting the channel catfish beta1 molecule has a molecular mass of approximately 130kDa, essentially the same as that for mammalian species. These results confirm the existence and expression of a beta1 gene in channel catfish, a species phylogenetically distant from mammals.

Molecular and functional characterization of channel catfish (Ictalurus punctatus) neutrophil collagenase.

Channel catfish (Ictalurus punctatus) neutrophils, like mammalian neutrophils, contain a variety of enzymes and lytic peptides that participate in pathogen destruction. We have identified and characterized from a channel catfish anterior kidney cDNA library a 1.6 kb cDNA that encodes for channel catfish neutrophil collagenase. The deduced amino acid sequence has a predicted molecular mass of 53 kDa. The putative catfish collagenase has nucleotide and amino acid homology of 51.4% and 45.1%, respectively, with human neutrophil collagenase and 50.4% and 47.1%, respectively, with mouse neutrophil collagenase. Certain regions of the molecule, including the cysteine switch and the putative zinc binding sites, were identical to those in the human and mouse genes. Polyclonal antiserum, prepared to the fusion protein, recognizes proteins from channel catfish neutrophil supernatants with molecular masses of approximately 63, 53 and 28 kDa. Supernatants from phorbol dibutyrate stimulated neutrophils were capable of degrading type I collagen. In addition, the polyclonal antiserum prevented the collagenase activity of the supernatants from stimulated catfish neutrophils; whereas, preimmune serum had no effect on collagenase activity of supernatants. Supernatants from unstimulated cells or the fusion protein did not possess the ability of degrading type I collagen. These results indicate that channel catfish neutrophil collagenases share molecular and functional features with mammalian neutrophil collagenase.

Magnetic resonance imaging of muscles in myotonic dystrophy.

The MR findings in 27 patients with myotonic dystrophy were compared with those observed in 11 patients with other muscular dystrophies: six with limb-girdle dystrophy, three with facioscapulohumeral muscular dystrophy and two with Becker-type muscular dystrophy. Clinical status was graded into 10 stages. The MR study was performed at the medium third of the thigh, with a slice thickness of 7.5 mm (TR: 750/TE: 25 for T1; TR: 2200/TE: 30/90 for DP/T2). Muscle signal intensity was evaluated with a four-point grading scale using subcutaneous fat as a reference. Statistical analysis was done using the Mann-Whitney-Wilcoxon's test and simple linear regression. In the myotonic dystrophy group, 81.4% of the patients showed an abnormal signal at the crural muscle level, adopting a semilunar shape around the anteroexternal side of the femur. The presence and intensity of this hyperintense signal correlated positively with the duration of disease (r = 0.54) and the clinical stage (r = 0.69). Of the 11 patients with other muscular dystrophies, only three (27.2%) showed hyperintense signal at the crural muscle level. MR imaging of patients with muscle disease may contribute to the in vivo study of muscular dystrophy, its differential diagnosis and the detection of asymptomatic patients.

Ion exchange chromatographic determination of olpadronate, phosphate, phosphite, chloride and methanesulfonic acid.

A method based on single column ion chromatography with UV detection was developed for purity testing and assay of monosodium olpadronate. The analyte aqueous solution is precipitated with methanol to enhance the impurities/olpadronate molar ratio, thus improving purity determination at trace levels. The resulting solution is injected into a standard chromatographic system with UV detector in indirect mode with a Waters IC Pak HR column using diluted nitric acid as the mobile phase. The method was fully validated according to ICH guidelines for the determination of phosphite, phosphate, chloride and methanesulfonic acid in olpadronate being suitable for purity testing and assay.

Isolation of viral hemorrhagic septicemia virus from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap.

Viral hemorrhagic septicemia virus (VHSV) was isolated from apparently healthy Greenland halibut Reinhardtius hippoglossoides caught in the Flemish Cap, a deep fishing ground in the North Atlantic Ocean in international waters near Newfoundland. The identity of the virus was confirmed by electron microscopy, immunodot, seroneutralization and reverse transcriptasepolymerase chain reaction. In the serology assays, all isolates reacted in the immunodot assay with a polyclonal antiserum against the European VHSV Type Strain F1, and were neutralized by the same antiserum, although most of the strains showed low or moderate neutralization titers. None of the isolates were detected by immunofluorescence using a specific monoclonal antibody against a nucleocapsid-related protein of VHSV F1. This is the first report of VHSV isolated from wild Greenland halibut, which represents a new host species for the virus, and it is also the first evidence of VHSV in a location close to the Atlantic coast of North America. This isolation indicates that VHSV is more widely distributed than has been thought, and appears to support a marine origin of this virus.

The tropicamide test in patients with dementia of Alzheimer type and frontotemporal dementia.

The tropicamide test was applied to 30 patients with probable Alzheimer's disease (pAD), 12 with frontotemporal dementia (FTD) and 46 healthy subjects. One drop of 0.01% tropicamide was instilled in one eye and one drop of saline solution in the other. The pupil diameter was measured with a Goldmann pupillometer in its basal condition and 10, 15, 20, 25, 30, 35, 45 and 55 minutes afterwards. The results do not show differences between pupil dilation observed in pAD and in FTD; in both groups, from the beginning of the test, the pupil dilated more than in healthy people. A high interindividual variability was observed. The best cutoff point is 38% of interpupillary difference at minute 25 (sensitivity = 63%, specificity = 80%). If we consider the prevalence of AD in a population over 40 years old to be 1.4%, the adjusted positive predictive value of the test would be 4.2%. According to these results, the test is not a true diagnostic marker of AD.

[Cytomorphological study of the cerebrospinal fluid. II. Leukemias and lymphomas (author's transl)]. / Citomorfología del líquido cefalorraquídeo. II. Leucemias y linfomas.

A cytological study of the cerebrospinal fluid from 25 patients with acute leukemias and five patients with malignant lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma) has been performed. All patients had clinical manifestations of central nervous system involvement at the time of lumbar puncture. Cytological abnormalities were observed in 13 out of the 30 samples examined (positivity: 44 percent). The group of 17 patients with lymphoblastic leukemia accounted for the highest cytological positivity (12 our of 17 cases --70.5 percent--). Cerebrospinal fluid from eight patients with acute myeloblastic leukemia and from four patients with Hodgkin's disease was normal. Numerous cell atypias were found in a case of lymphocytic lymphoma. The importance of systematic cytological study of the cerebrospinal fluid in patients with leukemia in order to prevent recurrences and to control intrathecal administration of cytostatics and/or radiotherapy is stressed.

[Cytomorphology of the cerebrospinal fluid. I. Tumors of the central nervous system (author's transl)]. / Citomorfología del líquido cefalorraquídeo. I. Tumores del sistema nervioso central.

A cytomorphological study of the cerebrospinal fluid corresponding to 42 patients with tumors of the central nervous system has been performed. The group of 31 patients with primary tumors accounted for an overall cytological positivity of 32.2 percent; according to the type of tumor cytological positivities were as follows: glioblastoma multiforme (44.4 percent); grade I/III astrocytoma (33.3 percent); oligodendroglioma (33.3 percent); ependymoma (25 percent), and meningioma (16.6 percent). In the group of 11 patients with metastatic tumors, neoplastic cells were observed in two cases (cytological positivity 18.1 percent). Cytological study of the cerebrospinal fluid is a useful diagnostic method for some primary and secondary neoplasms of the central nervous system.

[Computerized cerebral tomography in tuberous sclerosis. Study of 3 patients]. / Tomografía computarizada cerebral en la esclerosis tuberosa. Estudio de tres pacientes.

The findings on computerized axial tomography of the brain in three patients with tuberous sclerosis are reported. Numerous intracranial calcified nodules varying in size from a few millimeters to one centimeter, located preferentially below the ependyma of the lateral ventricles and determining filling defects in its lumen were found in all cases. Other calcified lesions were found in a subcortical location. One patient presented areas of low density throughout the hemispheric white matter, and another showed ventricular dilatation of the ventricles and basal cisternas. Computerized axial tomography is useful in the evaluation of patients with tuberous sclerosis because of its early demonstration of the characteristic intracranial lesions and detection of complications such as calcification of basal and cerebellar ganglia, hydrocephalus, and development of gliomas. Low density areas may correspond to foci of demyelinization. Furthermore, computerized axial tomography allows the demonstration of cerebral tuberosities in asymptomatic relatives, thus offsetting the inconveniences of pneumoencephalography.

[Detection of soluble interleukin-2 receptor in the serum of patients with non-Hodgkin's lymphoma]. / Determinación del receptor soluble de la interleucina 2 en el suero de pacientes con linfoma no hodgkiniano.

BACKGROUND: Patients with non-Hodgkin's lymphoma (NHL) have increased serum levels of soluble interleukin-2 receptor (sCD25). In this study the authors investigate: a) the value of sCD25, compared to other serum markers, as tumor marker, and b) the relationship of the sCD25 with the response to therapy and prognosis. PATIENTS AND METHODS: Serum interleukin-2 receptor (sCD25) levels were measured at diagnosis in 63 patients with NHL (low-grade lymphoma 30 and high-grade lymphoma 33). RESULTS: High levels of sCD25 were found in these patients compared to a control group (median 1,757 U/ml vs 385 U/ml; p < 0.0001). Significant differences were also found between the high-grade group and the low-grade group, as a whole and within the same Ann Arbor stage. sCD25 showed a correlation coefficient higher than other serum parameters (albumin, LDH, beta 2-microglobulin, uric acid, C-reactive protein) with Ann Arbor stage and with the number of involved lymph nodes or extralymphatic organs. In the high-grade NHL, the median of sCD25 (3,000 U/ml) separates patients with differences in the overall survival (p = 0.0138) and in percentage of complete remisions (p = 0.0079). All the patients with sCD25 < or = 3,000 U/ml reached the remision. The association sCD25 > 3,000 U/ml and albumin < 3.5 g/dl selected to 5 out of 6 patients who failed induction chemotherapy, and only 2 out of 22 who reached the remision. CONCLUSIONS: The sCD25 is the best serum factor for estimating tumor burden in NHL. sCD25 level isolates or associated with albumin provides prognostic information.

[Behçet's disease: report of ten cases and of a new clinical manifestation (portal vein thrombosis) (author's transl)]. / Enfermedad de Behçet: presentación de 10 casos y de una nueva manifestación clínica (trombosis portal).

Ten patients (seven males and three females) with the diagnostic criteria for Behçet's disease are reported. Symptoms began between ages 14 and 37 (mean age 27.9 years), and the time of evolution of the disease varied between one and 23 years (mean 5.7 years). Initial presentation was aphthous stomatitis (three cases), ocular inflammation (three cases), erythema nodosum (one case), arthritis (one case), thrombophlebitis (one case), and hemorrhagic proctocolitis (one case). During the course of the disease the commonest clinical manifestations were oral and genital ulcerations (100%) and ocular (70%), cutaneous (70%), articular (40%), neurological (40%), venous (40%), gastrointestinal (40%) and arterial (10%) involvement. Increased erythrocyte sedimentation rate was a constant feature in all patients. HLA-B5 antigen was determined in four patients, being positive in two of them. Cerebrospinal fluid alterations were present in the four patients with neurological involvement even though three of them never had meningeal symptoms. One patient developed portal hypertension secondary to portal vein thrombosis, and its clinical manifestations are described.

[Experience with pupil tropicamide test in Alzheimer's disease]. / Experiencia con el test pupilar de tropicamida en la enfermedad de Alzheimer.

Scinto et al (Science 1994; 266: 1051-4) observed that a single drop of 0.01% tropicamide elicited more than a 13% pupil dilatation in 19 individuals with probable or possible Alzheimer's disease (AD) and 3 cognitive abnormal elderly without dementia, but not in 30 out of 32 normal elderly and 3 out of 4 patients suffering from other dementias. It could be an easy and bloodless test to help in diagnosing AD. We have administered the same test to 10 patients with probable AD (NINCDS-ADRDA criteria) and twenty 40-90 year-old control subjects (10 of whom were sons or daughters of AD patients and 10 without a family history of dementia). The researcher does not know which group the subject belongs to. He administers a single drop of 0.01% tropicamide in eye and one drop of 0.45% normal saline solution in the other (without knowing the contents of either vial) and measures the pupil diameter by means of a Goldmann pupilometer in basal condition and 10, 15, 25, 30, 35, 45, and 55 minutes after. The results show that it is necessary to measure the pupils at least between the minutes 25 and 55 to detect tha maximum pupil dilation in every case. The cutoff point to consider the result positive must be located between 43 and 50%. If we establish the cutoff point in 50% of pupil dilation, 90% of AD patients and 35% of control subjects show a positive response. There was not a statistically significant difference between both control groups. Our results from this test show a sensitivity of 90% and a specificity of 65%. The positive responses of some control subjects may express a weak specificity, or perhaps they mean that we have a marker of the pre-clinical stage of the disease before us.

[A study of bilingual Galician-Castillian aphasic patients]. / Estudio de pacientes afásicos bilingües gallego-castellano.

INTRODUCTION: Aphasia in bilingual persons has some striking peculiarities. Most studies have been carried out in mono-lingual societies, where the second language has usually been learnt after immigration. In Galicia, linguistic closeness and simultaneous learning of Galician and Castilian languages permits the testing of neuropsychological theories conceived in other circumstances. OBJECTIVE: In aphasic bilingual Galician-Castilian patients we studied how this affected denomination, designation and translation, different modes of recovery, variables associated with differential affectation, the presence and influence of specific phenomena of aphasia in bilingual persons on changes in dominance and mixing of the languages. Material and methods. We selected 49 patients, 29 men and 20 women aged between 32 and 85 years old. We analyzed the variables associated with differential affectation: types of aphasia, size and site of the lesion, aetiology, age, educational level, and the presence and influence of phenomena specific to aphasia in bilingual persons. RESULTS: The test most affected was translation, to a lesser extent denomination, and finally designation. Capacity for recovery is greater in the dominant language. Translation recovers better than denomination and this better than designation. We found no changes in dominance, no selective loss of either language but have seen some interference in the non-dominant language. CONCLUSIONS: The two languages are seen to be recovered equally, as were other languages previously studied in aphasiology.

[A headache and ischemic infarction of middle cerebral artery: a prospective study of 100 patient]. / La cefalea en el infarto isquémico de la arteria cerebral media. Estudio prospectivo de 100 pacientes.

The frequency and clinical characteristics of headache were assessed in a prospective study carried out among 100 patients suffering from ischaemic infarct in the area of the middle cerebral artery. Headache was found in 28 out of the 100 patients studied and proved to be significantly more frequent in those infarcts located in the superficial branches (p < 0.001) and in the dominant hemisphere (p < 0.05). We found no relationship between the existence of headache and the type of infarct, its extension or level of neurological affectation. Severe headache preceded stroke in 28.5% of cases, was simultaneous in 57.1% and followed in 14.2%. The average intensity of headache was 2.14 +/- 1.69 headache units. It was bilateral in 46.4% of patients, generalized in 14.2%, unilateral on the side of the infarct in 28.5% and counterlateral in the remaining 10.7%.

[Migraine and histamine: determining histidine in plasma and cerebrospinal fluid during migraine attacks]. / Migraña e histamina: determinación de histidina en plasma y líquido cefalorraquídeo durante crisis de migraña.

We studied the plasma levels and cerebrospinal fluid (CSF) levels in twenty-nine patients suffering from migraine without aura and in eighteen others with aura. The samples were obtained during migraine attacks. We compared the results obtained with those from twenty-seven control subjects. Histidine levels in plasma were 1.18 +/- 0.51 mg/dl for the control group, 1.75 +/- 0.39 mg/dl in those suffering from migraine without aura and 2.07 +/- 0.64 mg/dl in those cases of migraine with aura. In both plasma and in cerebrospinal fluid concentrations of histidine were greater in patients with migraine than in controls (p < 0.001). Histidine is an amino acid precursor of histamine and its abnormalities may be demonstrative of the participation of this amine in migraine attacks, possibly within the state of neuronal hyperexcitability.