Rapid identification of Rhodococcus equi by a PCR assay targeting the choE gene

The actinomycete Rhodococcus equi is an important pathogen of horses and an emerging opportunistic pathogen of humans. Identification of R. equi by classical bacteriological techniques is sometimes difficult, and misclassification of an isolate is not uncommon. We report here on a specific PCR assay for the rapid and reliable identification of R. equi. It is based on the amplification of a fragment of the choE gene encoding cholesterol oxidase. The choE-based PCR was assessed by using a panel of strains comprising 132 isolates from different sources and of different geographical origins, all initially identified biochemically as R. equi, and 30 isolates of representative non-R. equi actinomycete species, including cholesterol oxidase producers. The expected 959-bp amplicon was observed only with R. equi isolates, as confirmed by sequencing of a variable region of the 16S RNA gene from a random sample of 20 PCR-positive isolates. All R. equi isolates gave a positive choE-based PCR result, which correlated with a high degree of conservation of the choE gene. Three of the 132 strains originally identified as R. equi were negative for the choE gene, and subsequent analysis of their 16S RNA gene sequences confirmed that they belonged to other bacterial species (Dietzia maris, Mycobacterium peregrinum, and Staphylococcus epidermidis). All non-R. equi isolates were negative by the choE-based PCR. ATCC 21387, the only known isolate of Brevibacterium sterolicum, gave a 959-bp amplicon whose DNA sequence was virtually identical to that of R. equi choE. Comparison of the 16S RNA genes indicated that ATCC 21387 should be considered an R. equi isolate.     

Cytochemistry and morphology of granulocytes of the caecilian <Emphasis Type="Italic">Siphonops annulatus</Emphasis> (Amphibia,Gymnophiona)

The caecilians (Amphibia, Gymnophiona) constitute one of the least known groups of terrestrial vertebrates because most species live underground in quite inaccessible environments. Siphonops annulatus is an exclusively fossorial species and is the most extensively distributed caecilian in South America. Little is known of this order concerning circulating granulocytes, including their morphological and cytochemical structure and ultrastructure. This paper is part of a project covering the study of granulocytes in representative species of the order Amphibia. Blood extensions were carried out and submitted to Leishman, Toluidine Blue, Periodic acid Schiff, Sirius Red and hydrogen o-toluidine peroxide methods. Part of the samples was prepared for conventional transmission electron microscopy. Among granular leukocytes, mature and immature neutrophils and eosinophils were identified, plus basophils. The most frequent granulocyte encountered in S. annulatus peripheral blood is the neutrophil. This is a cell with a hyper-segmented nucleus and with a very clear cytoplasm when compared to the eosinophil, which presents large cytoplasmic acidophilic granules. On the other hand, the basophils present basophilic and metachromatic granules. Glycogen was detected in the cytoplasm of the neutrophils and eosinophils, while basic protein rich in amino acids was observed in the eosinophil’s granules. Myeloperoxidase activity was detected in the cytoplasm of the neutrophils and eosinophils. Neutrophils were ultrastructurally detected with three types of small granules: eosinophils with large and small spherical granules and basophils with large spherical granules with lamellate structures.     

BCL-2--general considerations

BCL-2 protein is a oncoprotein considered to have an important role in the regulation of programmed cell death--apoptosis. The intracellular localization, the relation with other oncoproteins, the antioxidant function and the pathological implications are discussed.     

Clonal proliferation of cyclin D1-positive mantle lymphocytes in an asymptomatic patient: an early-stage event in the development or an indolent form of a mantle cell lymphoma?

Mantle cell lymphoma (MCL) is a B-cell neoplasm with a relatively aggressive clinical course. There is a very small subgroup of patients who present with atypical lymphocytes in peripheral blood, with or without lymphocytosis, lymphadenopathy, or splenomegaly, and with an indolent clinical course. They frequently show mutated IgV(H) genes and CD5 negativity. We report an asymptomatic elderly patient who presented with a single submandibular lymphadenopathy. The biopsy showed immunophenotype and t(11;14)(q13;q32) consistent with MCL. The abnormal lymphoid population was also detected in peripheral blood and bone marrow. The patient has remained asymptomatic for 5 years without receiving any therapy. It is uncertain whether these cases represent an early-stage event in the development or an indolent form of MCL. The existence of such asymptomatic patients with an indolent clinical course should induce a strict clinical judgment in terms of therapeutic decisions.     

Cross neutralization of SARS-CoV-2 omicron subvariants after repeated doses of COVID-19 mRNA vaccines

We have measured the humoral response to messenger RNA (mRNA) vaccines in COVID-19 naïve and convalescent individuals. Third doses of mRNA COVID-19 vaccines induced a significant increase in potency and breadth of neutralization against SARS-CoV-2 variants of concern (VoC) including Omicron subvariants BA.1, BA.2, and BA.2.12.1, that were cross-neutralized at comparable levels and less for BA.4/5. This booster effect was especially important in naïve individuals that only after the third dose achieved a level that was comparable with that of vaccinated COVID-19 convalescents except for BA.4/5. Avidity of RBD-binding antibodies was also significantly increased in naïve individuals after the third dose, indicating an association between affinity maturation and cross neutralization of VoC. These results suggest that at least three antigenic stimuli by infection or vaccination with ancestral SARS-CoV-2 sequences are required to induce high avidity cross-neutralizing antibodies. Nevertheless, the circulation of new subvariants such as BA.4/5 with partial resistance to neutralization will have to be closely monitored and eventually consider for future vaccine developments.     

1p36 deletion syndrome: Review and mapping with further characterization of the phenotype,a new cohort of 86 patients

Chromosome 1p36 deletion syndrome (1p36DS) is one of the most common terminal deletion syndromes (incidence between 1/5000 and 1/10,000 live births in the American population), due to a heterozygous deletion of part of the short arm of chromosome 1. The 1p36DS is characterized by typical craniofacial features, developmental delay/intellectual disability, hypotonia, epilepsy, cardiomyopathy/congenital heart defect, brain abnormalities, hearing loss, eyes/vision problem, and short stature. The aim of our study was to (1) evaluate the incidence of the 1p36DS in the French population compared to 22q11.2 deletion syndrome and trisomy 21; (2) review the postnatal phenotype related to microarray data, compared to previously publish prenatal data. Thanks to a collaboration with the ACLF (Association des Cytogénéticiens de Langue Française), we have collected data of 86 patients constituting, to the best of our knowledge, the second-largest cohort of 1p36DS patients in the literature. We estimated an average of at least 10 cases per year in France. 1p36DS seems to be much less frequent than 22q11.2 deletion syndrome and trisomy 21. Patients presented mainly dysmorphism, microcephaly, developmental delay/intellectual disability, hypotonia, epilepsy, brain malformations, behavioral disorders, cardiomyopathy, or cardiovascular malformations and, pre and/or postnatal growth retardation. Cardiac abnormalities, brain malformations, and epilepsy were more frequent in distal deletions, whereas microcephaly was more common in proximal deletions. Mapping and genotype–phenotype correlation allowed us to identify four critical regions responsible for intellectual disability. This study highlights some phenotypic variability, according to the deletion position, and helps to refine the phenotype of 1p36DS, allowing improved management and follow-up of patients.     

Enhanced survival of glioma bearing rats following boron neutron capture therapy with blood-brain barrier disruption and intracarotid injection of boronophenylalanine

Boronophenylalanine (BPA) has been used for boron neutron capture therapy (BNCT) of brain tumors in both experimental animals and humans. The purpose of the present study was to determine if the efficacy of BNCT could be enhanced by means of intracarotid (i.c.) injection of BPA with or without blood-brain barrier disruption (BBB-D) and neutron irradiation using a rat brain tumor model. For biodistribution studies, F98 glioma cells were implanted stereotactically into the brains of Fischer rats, and12 days later BBB-D was carried out by i.c. infusion of 25% mannitol (1.373 mOsmol/ml), followed immediately by i.c. administration of 300, 500 or 800 mg of BPA/kg body weight (b.w.). At the 500 mg dose a fourfold increase in tumor boron concentration (94.5 g/g) was seen at 2.5 hours after BBB-D, compared to 20.8 g/g in i.v. injected animals. The best composite tumor to normal tissue ratios were observed at 2.5 hours after BBB-D, at which time the tumor: blood (T: Bl) ratio was10.9, and the tumor: brain (T: Br) ratio was 7.5, compared to 3.2 and 5.0 respectively for i.v. injected rats. In contrast, animals that had received i.c. BPA without BBB-D had T: Bl and T: Br ratios of 8.5 and 5.9, respectively, and the tumor boron concentration was 42.7g/g. For therapy experiments, initiated 14 days after intracerebral implantation of F98 glioma cells, 500 mg/kg b.w. of BPA were administered i.v. or i.c. with or without BBB-D, and the animals were irradiated 2.5 hourslater at the Brookhaven Medical Research Reactor with a collimated beam of thermal neutrons delivered to the head. The mean survival time for untreated control rats was 24 ± 3 days, 30 ± 2 days for irradiated controls, 37 ± 3 days for those receiving i.v. BPA, 52 ± 15 days for rats receiving i.c. BPA without BBB-D, and 95 ± 95 days for BBB-D followed by i.c. BPA and BNCT. The latter group had a 246% increase in life span (ILS) compared to untreated controls and a 124% ILS compared to that of i.v. injected animals. These survival data are the best ever obtained with the F98 glioma model and suggest that i.c. administration of BPA with or without BBB-D may be useful as a means to increase the efficacy of BNCT.     

Tumor proliferative activity and response to first-line chemotherapy in advanced breast carcinoma

Summary The relationship between tumor proliferative activity and response to first-line chemotherapy and survival was investigated in 76 advanced breast cancer patients. Proliferative activity was determined by means of Ki-67 immunohistologic staining on primary tumors (55 patients) or at the relapse site (21 patients), and was classified as low ( 25% of stained cells) or high (> 25% of stained cells). The usual WHO response criteria were used. The median duration of follow-up was 18 months (range 3–58).Forty-seven patients (62%) had tumors with low, and 29 (38%) had tumors with a high rate of proliferative activity. The two groups were well balanced in terms of important variables such as disease-free survival, performance status, age, menopausal status, and the type of first-line chemotherapy (anthracycline-based regimens versus cyclophosphamide-methotrexate-5-fluorouracil). The estrogen receptor (ER) content, measured by means of immunohistochemical assay, was markedly different in the two groups, with 27/47 tumors with low proliferative activity (57%) and 6/29 with high-proliferative activity (21%) being ER positive ( 45% of stained cells) (p = 0.003). Moreover, a significant difference in the metastatic pattern was also evident, with a higher incidence of bone and a lower incidence of soft tissue metastases in the group of patients with tumors with low proliferative activity (p = 0.004). Overall, 10/47 responses (21%: PR = 7, and CR = 3) were observed in the group with a low rate of proliferative activity, versus 14/29 (48%: PR = 9, and CR = 5) in the group with highly proliferative tumors, the difference being statistically significant (p = 0.03). When a multivariate analy-sis was performed, the only factor that retained independent prognostic significance was the predominant site of disease, particularly soft tissues (p = 0.003). Despite the difference in response rate, when survival analysis was performed according to the Kaplan-Meier method, no significant difference was observed in the two groups, but when the analysis was limited to responsive patients, the median survival observed in those with a low and those with a high rate of proliferation was 35 and 19 months respectively (p = 0.02). The same results were obtained when multivariate survival analysis was carried out using Cox's regression model. These data suggest that there is a link between tumor proliferative activity and response to chemotherapy in advanced breast cancer, and may indicate the need to use more intensive treatments in selected patients with highly proliferative tumors.Presented in part at the Annual Meeting of the American Society of Clinical Oncology, May 14–17, 1994, Dallas, TX, USA     

The influence of cardiac cholinergic activation on the induction and maintenace of ventricular fibrillation

Summary The influence of cardiac cholinergic activation was studied in rats and cats on the induction and maintenance of ventricular fibrillation (VF). Acetylcholine (ACH 2–25 g/kg), in doses which did not cause bradycardia or hypotension, induced appearance of spontaneous VF (duration 2–60 sec.) in 9/20 rats which have a high sympathetic autoregulation and in 3/6 cats only, 20–40 secs after the latter had been given adrenaline. ACh (10–45 g/kg) and methacholine (10–40g/kg) also significantly prolonged the fibrillatory period induced electrically in cats and rats with and without atrial or ventricular pacing. The induction or prolongation of VF did not occur when higher doses of ACh (50–100 g/kg) were given to rats. The influence of moderate amounts of cholinergic agents on the heart may be due to localised effects resulting in asynchronous activity. Alternatively, they may produce a discharge of multiple ectopic pacemakers or a disturbance in impulse conduction. Higher doses of ACh depress the S-A and ventricular ectopic activity node thereby decreasing the probability of inducing VF.It is concluded that under conditions of raised cardiac adrenergic activity, a moderate increase in cholinergic influence can both induce and prolong VF. The relevance of these findings to the sudden infant death syndrome is discussed.     

SIALYL-Tn ANTIGEN DISTRIBUTION IN HELICOBACTER PYLORI CHRONIC GASTRITIS IN CHILDREN: AN IMMUNOHISTOCHEMICAL STUDY

Sialyl-Tn antigen (STn) is a mucin-type carbohydrate normally present in goblet cells of small and large bowel. STn expression has been demonstrated to occur in complete and incomplete intestinal metaplasia as well as in many carcinomas but in no normal gastric cell. The aim of our present study was to evaluate the distribution of STn in Helicobacter pylori chronic gastritis (HpCG) of pediatric patients. Eighteen gastric biopsies from 15 children (mean age: 11.5 years) with HpCG, 9 gastric biopsies from 9 children without H. pylori infection, and 1 heterotopic gastric mucosa in Meckel's diverticulum were immunostained using the anti-STn antibody STn1 (18/18), NCL-MUC-1 (7/18), and NCL-MUC-2 (18/18) antibodies. Also, sulfated mucosubstances were investigated with the Alcian Blue-Periodic Acid Schiff (AB-PAS), pH 1.0 stain. Although with different intensity (weak in 5/18, moderate 9/18, and intense 4/18) all cases with HpCG exhibited STn immunoreactivity. The expression of STn was found to be located mainly to the supranuclear region of the epithelial cells at the foveolae and glandular necks, with occasional cells showing diffuse cytoplasmic staining. When reactivity was intense, it was for the most part found in the cells at the neck of the glands. The mucus out of the luminal border above the positive cells was usually also stained. MUC-1 was negative (2/7) or weakly positive (5/7) in a few surface mucous cells. MUC-2 was negative (16/18) or occasionally detected in some foveolar and surface cells (2/18). AB-PAS pH 1.0 revealed the presence of sulfomucins in the cytoplasm of isolated cells of gastric pits and glands of most cases (11/15). None of these findings was observed in the control group. We conclude that STn can be identified in gastric cells of pediatric patients with HpCG and that this does not correlate with other mucosubtances markers. Thefindings could indicate that minimal intestinal metaplasia takes place in children with HpCG.