Histogenesis of cardiac myxomas. An immunohistochemical study of 19 cases, including one with glandular structures, and review of the literature

To elucidate the histogenesis of cardiac myxomas, the literature has been reviewed, including all previous immunohistochemical studies, and an extensive immunohistochemical study of 19 cardiac myxomas has been undertaken with antibodies to factor VIII, desmin, vimentin, myoglobin, cytokeratin (CAM 5.2 and AE1/AE3), and S100 protein. In all cases, vimentin stained endothelial as well as "myxoma" (stromal) cells. In contrast, factor VIII only stained endothelial cells. In 16 cases, desmin stained cells in the vascular structures; in 5 cases, desmin stained myxoma cells. In 6 cases, S100 protein stained the myxoma cells strongly. Myoglobin was absent in all cases. In 1 case, CAM 5.2 and AE1/AE3 stained glandular structures. The results indicate the following: that most cardiac myxomas are true neoplasms derived from "embryonal rests"; that the various mesenchymal cells found in cardiac myxomas (myxoma cells, endothelial cells, smooth-muscle cells, fibroblasts, myofibroblasts, and chondroid cells), express differentiation, not histogenesis; that, apparently, only the myxoma cells are neoplastic; and that the glandular structures infrequently found in cardiac myxomas originate from entrapped foregut rests.     

Impaired phytohaemagglutinin-induced cytotoxicity in vitro of lymphocytes from patients with Hodgkin''s disease or chronic lymphatic leukaemia

Lymphocytes from healthy, unsensitized donors damage allogeneic tissue culture target cells in the presence of phytohaemagglutinin (PHA). In this investigation, blood lymphocytes from eleven patients with clinically active Hodgkin's disease and from eight patients with chronic lymphatic leukaemia (CLL) were tested in vitro for PHA-induced cytotoxicity to Chang cells. Blastoid transformation and DNA-synthesis in the presence of PHA were studied in parallel experiments. The cytotoxicity of the patients' lymphocytes was significantly lower than that of lymphocytes from healthy donors. Synthesis of DNA and blastoid transformation was subnormal in all cases of CLL and in most cases with Hodgkin's disease. A good correlation between cytotoxicity and stimulation by PHA was observed in most cases.

The incidence of positive skin reactions to tuberculin was high in the CLL-group and low in the Hodgkin group. If it is assumed that the pool of immunologically competent cells was normal in the CLL-patients, this would account for their intact cellular reactivity in vivo. In contrast, the high proportion of leukaemic cells could be expected to mask the reactivity of the normal CLL-lymphocytes in vitro. The weak in vitro reactivity of the lymphocytes in Hodgkin's disease is in line with the view that deficient lymphocytes are present in this disease and suggests that the patients' pool of immunologically competent cells is reduced. This could also contribute to the anergic state seen in these patients.

     

Discrimination of human pathogenic subspecies of Francisella tularensis by using restriction fragment length polymorphism

We describe the use of two insertion sequence elements (ISFtu1 and ISFtu2) in Francisella tularensis to type strains by restriction fragment length polymorphism (RFLP). The RFLP profiles of 17 epidemiologically unrelated isolates were determined and compared. Our results showed that RFLP profiles can be used to assign F. tularensis strains into five main groups corresponding to strains of F. tularensis subsp. tularensis, F. tularensis strain ATCC 6223, strains of F. tularensis subsp. holarctica, strains of F. tularensis subsp. holarctica from Japan, and F. tularensis subsp. mediaasiatica. The results confirm the genetic identities of these subspecies and also support the suggestion that strains of F. tularensis subsp. holarctica from Japan should be considered members of a separate biovar. These findings should support future studies to determine the genetic differences between strains of F. tularensis at the whole-genome level.     

Accuracy and choice of procedures in 24-hour oesophageal pH monitoring with monocrystalline antimony electrodes

In 24 h pH monitoring, the evaluation is dependent on the absolute accuracy of the pH measurements. Several sources of error exist, such as the chemical composition of calibration buffers and reference electrode gel and the effect of temperature on both the pH and the reference electrodes. We investigated the magnitude of these errors for the monocrystalline antimony electrode. Similar analysis applies to other types of pH electrodes. The errors we found are important when choosing a calibration procedure. We recommend a calibration procedure in which the pH and reference electrodes are both put in a beaker with the calibration buffers prior to and after the 24 h measurements. The calibration buffers and the electrode gel should have a specially selected ion composition where, for example, the Cl-ion concentration is critical. Corrections for differences in temperature between the calibration and the in situ measurements must be added. The pH measurements can be checked by performing in situ calibration.     

In Vitro and In Vivo Interactions of Haemophilus ducreyi with Host Phagocytes

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10⁶ CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted throughout the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.     

Multilocus sequence typing of methicillin-resistant Staphylococcus aureus from an area of low endemicity by real-time PCR

A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCCmec) type identified 27 different genotypes, and type IV SCCmec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCCmec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCCmec-typing results when investigating the origins of MRSA.     

Improved cell depletion in a panning technique using covalent binding of immunoglobulins to surface modified polystyrene dishes

A method for the chemical modification of plastic surfaces permits covalent binding of proteins and we have used this method in the development of an efficient panning technique. Thus, human peripheral T lymphocytes coated with mouse monoclonal antibodies directed against the CD4 marker may be selectively and reproducibly removed from a lymphocyte population by a short incubation in modified plastic dishes coated with rabbit anti-mouse IgG antibody. Due to the higher protein binding capacity of the dishes the use of the IgG fraction of the coating antibody was sufficient for optimal and reproducible results. In contrast, control dishes with passively adsorbed antibody required an affinity-purified fraction and even then were less efficient.     

Use of multiple PCR primer sets for optimal detection of human papillomavirus.

Using multiple PCR primer sets, we tried to optimize the detection of human papillomavirus (HPV) in DNA samples isolated from 361 frozen biopsy specimens from patients with invasive cervical carcinomas. The HPVs detected were placed into three distinct groups, including group I/Inex at Telelab (Skien, Norway) and group Ineg and group II at the Norwegian Radium Hospital (Oslo, Norway). The consensus primer sets were Oli-1b-oli-2i, My09-My11, Gp5-Gp6, and Gp(5+)-Gp6+ from the HPV L1 gene and CpI-CpIIG from the E1 gene. Using these consensus primers together with the type-specific primers from E6-E7, we found that 355 patients (98%) were HPV positive. Type-specific primers for HPV types 11, 16, 18, 31, 33, and 35 detected more HPV-infected patients than the most sensitive consensus primer set, while the three consensus primer sets My, Gp/Gp+, and Cp together detected more HPV-positive patients than the type-specific primers. Testing of sensitivity of the PCR with SiHa cells serially diluted in lymphocytes (HPV-negative cells) indicated a detection limit of 6,300 HPV type 16 DNA copies with consensus primers (My, Gp+, and Cp) and 126 original HPV type 16 DNA copies with type-specific primers. Comparison of the amplification results for consensus L1 primers and type-specific E6-E7 primers indicated the presence of L1 deletions in 23 of 56 samples. The conclusion is that in PCR detection systems, multiple consensus primers and type-specific primers should be used in order to detect all patients harboring HPV.     

Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro

The mammalian tooth pulp becomes innervated by nociceptive and sympathetic axons relatively late during development, when part of the root has formed. In the adult, regenerating axons from an injured tooth nerve or sprouting axons from uninjured nerves in the vicinity rapidly reinnervate denervated tooth pulps. These observations indicate that tooth pulp tissue can use molecular factors to attract pulpal axons from local nerve trunks. The present study examines the hypothesis that these factors include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF). Explants of trigeminal ganglia from neonatal rat pups showed a distinct neurite outgrowth when co-cultured with pulpal explants collected from molar teeth of 12-day old pups, or after application of a pulpal extract. Control cultures, containing single ganglionic explants, or explants co-cultured with heat-treated pulpal tissue, exhibited a sparse neurite outgrowth. Exogenous NGF and/or GDNF, but not exogenous BDNF, stimulated neurite outgrowth from ganglionic explants. Unexpectedly, application of antibodies against NGF, BDNF and/or GDNF to co-cultures of ganglionic and pulpal explants did not inhibit neuritogenesis. Control experiments showed that IgG molecules readily penetrate the gel used for culture and that even very high concentrations of NGF and GDNF antibodies in combination failed to block neurite growth. On the basis of these data we suggest that other as yet unknown neurite-promoting factors might be present and active in TG/pulpal co-cultures.     

Monosomy and trisomy of 15q24→qter in a family with a translocation t(6;15)(P25;q24)

A child with multiple anomalies, including growth retardation, a left-sided diaphragmatic hernia with lung hypoplasia, and cerebral malformations is described. Cytogenetic investigation demonstrated a deletion of the distal part of one chromosome 15, del(15)(q24qter), an aberration not previously described. Family studies revealed that the mother had a balanced translocation, t(6;15)(p25;q24). Two of her subsequent pregnancies resulted in abortions after prenatal diagnosis: one fetus was trisomic for 15q24→qter, while the other had monosomy 15q24→qter and a left-sided diaphragmatic hernia similar to the first child.