Dilution assessment of cervicovaginal secretions obtained by vaginal washing for immunological assays.

Local immunological defense mechanisms in the cervicovaginal mucosa currently remain incompletely defined, especially from a quantitative point of view. Addition of an inert substance, lithium chloride (LiCl), into the washing buffer used to carry out the vaginal washing for collecting cervicovaginal secretions and measurement of its concentration with a flame absorption spectrophotometer, before and after the specimen is sampled, permits the quantification of the volume of cervicovaginal secretions collected and the approximation of the dilution factor of a soluble component introduced by the washing. Lithium, at a concentration of 10 mM, gives the best precision of measurement and has no effect on the results of the immunoassays. In a population of 27 nonpregnant women (age range, 18 to 45 years), the volume of cervicovaginal secretions collected by vaginal washing with 3 ml of LiCl-phosphate-buffered saline was 12% +/- 3.2% (mean +/- standard deviation) of the total volume and showed large interindividual variations (range, 5.6 to 18.8%); the mean dilution factor of a soluble component from the vaginal secretions was 9.9% +/- 2.8% (range, 6.3 to 18.8%). According to the date of the menstrual cycle, the mean volume of collected cervicovaginal secretions was significantly increased in the luteal phase in comparison with the follicular phase; conversely, the mean dilution factor of a soluble component was more important in the follicular than in the luteal phase. These features strengthen the need to quantify accurately the dilution factor introduced by vaginal washing when studying cervicovaginal immunity.     

Ovarian stimulation for in-vitro fertilization combining administration of gonadotrophins and blockade of the pituitary with D-Trp6-LHRH microcapsules: pilot studies with two protocols

In women undergoing in-vitro fertilization and embryo transfer(TVF-ET), a total of 408IVF cycles were stimulated using humanmenopausal gonadotrophin (HMG) or pure follicle stimulatinghormone (FSH) plus HMG in combination with a single injectionof D-Trp⁶-LHRH microcapsules in order to enhance the ovarianresponse to gonadotrophins and to avoid spontaneous LH surges.Sixty-seven pregnancies were achieved. Two protocols were employed.In protocol 1 (‘blocking protocol’, n = 268), thepituitary was first inhibited with a full dose (3.75 mg) ofD-Trp⁶-LHRH in microcapsules and ovarian stimulation was startedafter the hypogonadotrophic hypogonadal state was ascertained(Ej >50 pg/ml). In protocol 2 (‘flareup protocol’,n = 140), the treatment with D–Trp⁶LHRH microcapsules(half-dose = 1.80 mg) and the ovarian stimulation with gonadotrophinswere started at the same time. Higher doses of gonadotrophinswere needed (39.5 11.2 ampoules FSH and/or HMG) in protocol1, in which the pituitary was blocked prior to and during thestimulation, than in protocol 2 (209 ampoules) where the exogenousgonadotrophin stimulation appeared to be augmented by the initialagonistic effect of the injection of D-Trp⁶LHRH microcapsules.In patients with purely tubal infertility, under 38 years oldand no male factor, the results obtained with protocols 1 and2 were similar in terms of pregnancy rate per cycle or per embryotransfer: 22.6 versus 20.5% and 28.3 versus 27.4%, respectively.However, considering the cost benefit, ‘flare-up’protocols appeared to be a better choice and could be recommended.     

Distribution of nocardia species in clinical samples and their routine rapid identification in the laboratory

Eighty-six Nocardia strains isolated from clinical samples in Belgium were identified by 16S rRNA gene sequencing. Eighty-three (96%) strains belonged to only six Nocardia species: N. farcinica (38 [44%]), N. nova (19 [22%]), N. cyriacigeorgica (13 [15%]), N. brasiliensis (6 [6.9%]), N. abscessus (5 [5.8%]), and N. paucivorans (2 [2.3%]). A gallery of nine conventional and enzymatic tests was developed for the rapid identification of the most common species isolated during this survey. Pyrrolidonyl aminopeptidase, γ-glutamyl aminopeptidase, α-mannosidase, and α-glucosidase were found to be highly discriminating and could be used to develop an identification scheme.     

Requirement for tumor necrosis factor receptor 2 expression on vascular cells to induce experimental cerebral malaria

Using tumor necrosis factor receptor type 2 (TNFR2)-deficient mice and generating bone marrow chimeras which express TNFR2 on either hematopoietic or nonhematopoietic cells, we demonstrated the requirement for TNFR2 expression on tissue cells to induce lethal cerebral malaria. Thus, TNFR2 on the brain vasculature mediates tumor necrosis factor-induced neurovascular lesions in experimental cerebral malaria.     

Bacteremia due to Moraxella atlantae in a cancer patient

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as M. atlantae (formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that M. atlantae, M. lincolnii, and M. osloensis might constitute three separate genera within the MORAXELLACEAE: After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.     

ALK Expression Defines a Distinct Group of T/Null Lymphomas (“ALK Lymphomas”) with a Wide Morphological Spectrum

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffin sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma “common” type (75%), “lymphohistiocytic” (10%), “small cell” (8.3%), “giant cell” (3.3%), and “Hodgkin’s like” (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin’s-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and “Hodgkin’s-like” features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype (“ALK lymphomas”), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.     

A monoclonal antibody against a new differentiation antigen of thymocytes

B14-2-14 is a monoclonal cytotoxic IgM antibody which reacts with thymocytes of all mouse strains tested. The fraction of positive cells (by visual immunofluorescence) varies between strains from about 25-45% in A.CA to 65-85% in C57BL/6, and high levels are dominant in F1 hybrids. In the periphery, the antigen is found on a few percent of lymph node and not on splenic T cells, and it is absent in nude mice. Among thymocytes, the distribution of the B14 determinant largely overlaps with that of the TL antigen and of molecules binding peanut agglutinin. The B14 antibody reacts only minimally with hydrocortisone-resistant thymus cells. Biochemical analysis shows that B14 antibody, anti-TL antibody and peanut agglutinin bind to separate molecules. The target of the B14 antibody may be either an immature, thymic form of Thy-1, or another molecule associated with it. Two polypeptides, of 40 and 35 kDa are precipitated by both B14 and anti-Thy-1 antibodies from biosynthetically labeled thymus cell lysates, and two others, of 27 and 17 kDa, from surface-iodinated thymus cell preparations. B14-2-14 offers an additional method for identification and selection of thymocytes at different stages of differentiation, and should also be useful for studies of the Thy-1 antigen.     

Inflammatory malignant fibrous histiocytomas and dedifferentiated liposarcomas: histological review, genomic profile, and MDM2 and CDK4 status favour a single entity

Inflammatory malignant fibrous histiocytoma (inflammatory MFH) is a very rare tumour that occurs most often in the retroperitoneum. So far, it has been considered to be a special subtype of MFH. As it is now widely accepted that most retroperitoneal pleomorphic MFHs are dedifferentiated liposarcomas, the present study compared histological features, genomic profile (CGH analysis), and MDM2 and CDK4 status (immunohistochemistry, FISH, and quantitative PCR) in inflammatory MFHs from 12 patients and dedifferentiated liposarcomas that had an inflammatory MFH component from eight patients. Metaphase cytogenetic and FISH analyses were also performed on one inflammatory MFH. Histological review showed areas of well-differentiated liposarcoma in nine inflammatory MFHs. CGH analysis showed 12q13-15 amplification or gain in six of seven inflammatory MFHs and in seven of seven dedifferentiated liposarcomas. Immunohistochemistry showed positivity of tumour cells for MDM2 in every tumour in both groups and for CDK4 in ten and seven inflammatory MFHs and dedifferentiated liposarcomas, respectively. Metaphase cytogenetic and FISH analysis performed on one inflammatory MFH showed the presence of a supernumerary large marker chromosome and ring chromosome with high-level amplification of both MDM2 and CDK4 genes. FISH analysis on paraffin wax-embedded sections showed amplifications of MDM2 and CDK4 in seven of seven inflammatory MFHs and in seven of seven dedifferentiated liposarcomas. Quantitative PCR showed amplification of MDM2 in six and of CDK4 in seven of nine inflammatory MFHs. In conclusion, this study strongly suggests that most so-called inflammatory MFHs are dedifferentiated liposarcomas.     

Genotypic analysis of sequential genital herpes simplex virus type 1 (HSV-1) isolates of patients with recurrent HSV-1 associated genital herpes

Clinical recurrences of Herpes simplex virus type 1 (HSV-1)-associated genital herpes are thought to be caused by reactivation of latent endogenous HSV-1. However, the possibility of reinfection with exogenous HSV-1 cannot be excluded. This study aimed to determine the incidence of genital HSV-1 superinfection in patients by investigating the genotype of sequential HSV-1 isolates obtained from the same anatomical site of patients with clinical recurrences of genital HSV-1 recurrent genital herpes. Sequential genital HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1 and US12. Whereas the sequential HSV-1 isolates in 11 of the 13 patients studied had the same genotypes, the sequential isolates of 2 patients showed a different genotype. The data suggest that HSV-1-induced recurrent genital herpes can be associated with genital reinfection with an exogenous HSV-1 strain.