The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes.

Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia. In order to investigate whether the major surface antigen (MSG) of human P. carinii is capable of eliciting the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG. MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0.01). In a similar fashion, MSG elicited release of TNF-alpha. Initial increases were also seen at 4 h, and at 20 h, TNF-alpha levels reached 6.4 +/- 1.1 ng/ml, compared to 0.08 +/- 0.01 ng/ml for control cultures (P < 0.01). A concentration-dependent increase in IL-8 and TNF-alpha secretion was observed at 20 h with 0.2 to 5 microg of MSG/ml (P < 0.01). Secretion of IL-8 and TNF-alpha from MSG-stimulated monocytes at 20 h was inhibited by 60 and 86%, respectively, after coincubation with soluble yeast mannan (P = 0.01). With an RNase protection assay, increases in steady-state mRNA levels for IL-8 and TNF-alpha were detectable at 4 h. These data show that recognition of MSG by monocytes involves a mannose-mediated mechanism and results in the release of the proinflammatory cytokines IL-8 and TNF-alpha.     

Non-specific induction of cytotoxicity in normal human lymphocytes in vitro: studies of mechanism and specificity of the reaction

The cytotoxic effect of non-immunized human lymphocytes was investigated in a system where the lymphocytes were applied to fibroblast monolayers of different genotypes. Non-immunized lymphocytes were not cytotoxic, disregarding the target cell genotype provided that the lymphocyte suspensions were free from contaminating granulocytes. By adding phytohaemagglutinin (PHA) to the culture, the lymphocytes became strongly cytotoxic and exerted their effect already after 4–8 hr. Cytotoxicity was shown to develop independently of other expressions of PHA-stimulation of the lymphocyte, such as RNA-, protein- and DNA-syntheses and morphological transformation. Living lymphocytes were required for cytotoxicity to occur and heating the lymphocytes to 48·5°C, ultrasound disintegration or freezing-thawing abolished their ability to damage the target cells. The PHA-induced cytotoxicity was equally expressed on allogeneic and autochthonous fibroblasts. Analogous results were obtained when the lymphocytes were stimulated by streptolysin O or anti-lymphocyte serum. The results suggest that expression of cytotoxicity is an immunologically non-specific process, caused by stimulated lymphocytes. When the lymphocytes have acquired a cytotoxic potential they do not discriminate between the target cell genotype or the event triggering lymphocyte cytotoxicity. The specificity of the cellular immune reactions is probably confined to the immunological recognition step initiating the cytotoxic potential. This recognition step is by-passed if the lymphocytes are stimulated by PHA or other non-specific stimulators.     

Inflammatory response in bronchoalveolar lavage fluid after inhaling histamine

To examine the influence of the histamine chloride challenge test on the bronchoalveolar lavage cell population, lavage fluid from 15 subjects was collected 24 h after the histamine test, and was compared with the lavage fluid from a reference group of 25 subjects. Inhaled histamine is commonly used to quantitate non-specific bronchial responsiveness. Increase in airway responsiveness after exposure to ozone or allergen is associated with airway inflammation. Bronchoalveolar lavage, has therefore become a valuable tool in the study of bronchoalveolar cells and mediators in subjects with asthma and bronchial hyperresponsiveness. The total cell number and differential cell counts in bronchoalveolar lavage fluid 24 h after inhalation challenge test with histamine-chloride were studied. There was a significant increase in lymphocytes, mast cells and neutrophils after histamine test. The conclusion was that inhaled histamine-chloride can induce an inflammatory cell response in the lung. Thus the histamine-chloride test should not be performed before bronchoalveolar lavage.     

YKL-40 Is Elevated in Cerebrospinal Fluid from Patients with Purulent Meningitis

YKL-40, a member of the family 18 glycosyl hydrolases, is secreted by activated neutrophils and macrophages. It is a growth factor for connective tissue cells and a potent migration factor for endothelial cells and may function in inflammation and tissue remodeling. YKL-40 was determined in 134 cerebrospinal fluid (CSF) samples taken on admission from patients suspected of having meningitis (48 with purulent meningitis, 49 with lymphocytic meningitis, 5 with encephalitis, and 32 without evidence of meningitis). YKL-40 levels in CSF were significantly higher in patients with purulent meningitis (median, 663 μg/liter [range, 20 to 8,960]) and encephalitis (5,430 μg/liter [620 to 11,600]) than in patients with lymphocytic meningitis (137 μg/liter [41 to 1,865]) or patients without meningitis (167 μg/liter [24 to 630]) (Kruskal-Wallis and Dunn multiple comparison tests, P < 0.001). CSF YKL-40 levels were also determined for 26 patients with purulent meningitis having a repuncture, and patients who died (n = 5) had significantly higher YKL-40 levels than patients who survived (n = 21) (2,100 μg/liter [1,160 to 7,050] versus 885 μg/liter [192 to 15,400], respectively; Mann-Whitney test, P = 0.018). YKL-40 was most likely locally produced, since patients with infections of the central nervous system had CSF YKL-40 levels that were at least 10-fold higher than the corresponding levels in serum (2,033 μg/liter [470 to 11,600] versus 80 μg/liter [19 to 195]). The CSF neopterin level was the biochemical parameter in CSF and blood that correlated best with CSF YKL-40 levels, indicating that YKL-40 may be produced by activated macrophages within the central nervous system. In conclusion, high levels of YKL-40 in CSF are found in patients with purulent meningitis.     

Inhibition of acetylcholine release in guinea pig ileum by adenosine.

The effect of adenosine on cholinergic neuroeffector transmission was studied in the isolated guinea pig ileum. Adenosine caused a dose-dependent and inverse frequency-dependent inhibition of contraction responses to transmural nerve stimulation. Blockade of adrenergic neurotransmission did not alter the inhibitory effect of adenosine. Adenosine also inhibited contraction responses to serotonin, angiotensin and high potassium, but not the responses to acetylcholine, histamine or direct electrical stimulation of the smooth muscle cells. Adenosine had little effect on basal outflow of acetylcholine but inhibited markedly and reversibly the release of acetylcholine induced by nerve stimulation. Acetylcholine was determined with gas chromatography-mass spectrometry. The results provide direct evidence that adenosine inhibits cholinergic neuroeffector transmission in the gut by a prejunctional action on acetylcholine release. This may be of functional importance since adenine compounds are released during stimulation of intestinal nerves.     

Effects of variations in calcium concentration on parathyroid morphology in vitro.

Morphologic examinations, including the use of the potassium pyroantimonate-technique and x-ray analysis for ultrastructural localization of cations, were carried out on parathyroid glands from Mongolian gerbils cultured for 2 to 6 days. At a high calcium concentration most (suppressed) chief cells were characterized by a moderate or high cytoplasmic density, inconspicuous endoplasmic reticulum and Golgi complex, numerous often enlarged mitochondria, and the occasional occurrence of accumulations of secretory granules, storage granules, and lysosomal bodies, whereas other (atrophic) cells were smaller and still more dense. At a low concentration of calcium some (stimulated) chief cells were characterized by a low cytoplasmic density, normal or structurally altered mitochondria, and smooth surface vacuoles occasionally connected with mitochondria, whereas the main features of other (active) chief cells were a moderate or low cytoplasmic density and a prominent endoplasmic reticulum and Golgi complex. Calcium-containing precipitates were found both inter- and intracellularly; the latter were mainly localized to nuclei, mitochondria, and rough surfaced vacuoles, and in stimulated cells also to the smooth surfaced vacuoles and the cytosol. Degenerative changes were seen, mainly in the suppressed cells; massive calcium loading in mitochondria seemed to precede degeneration and necrosis of whole cells. The findings indicate that clear differences exist between the different chief cell variants both as to the distribution of calcium-containing precipitates and to the structural appearance, and that these differences are influenced by the ambient calcium concentration. Suppressed cells occasionally seem to accumulate hormone which later may undergo lysosomal digestion. Calcium might be accumulated intracellularly, mainly in mitochondria of suppressed cells.     

Helicobacter pylori-specific CD4+ CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals

Helicobacter pylori colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of H. pylori-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to H. pylori in H. pylori-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4+ CD25(high) T cells during infection. The stimulation of CD4+ peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of H. pylori resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4+ CD25(high) regulatory T cells or the addition of interleukin 2. Furthermore, CD4+ CD25(high) T cells suppressed H. pylori-induced responses in cocultures with CD25(low/-) cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was H. pylori specific. In conclusion, we have shown that H. pylori-infected individuals have impaired memory CD4+ T-cell responses to H. pylori that are linked to the presence of H. pylori-specific regulatory T cells that actively suppress the responses.     

A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme

A total of 364 consecutive patients requesting in-vitro fertilization (IVF) treatment were divided randomly into two groups. In the first group, two embryos in the original IVF cycle were allowed to divide prior to transfer, with any remaining embryos being cryopreserved at the pronucleate (PN) stage. In the second group, all the embryos were allowed to divide to the early cleavage (EC) stage, and the best two replaced; any suitable remaining embryos were frozen at the 2- to 4- cell stage. A total of 134 cycles (36.8%) fulfilled the study criteria for a fresh embryo replacement and supernumerary embryos cryopreserved. In the PN group, 72 out of 182 (39.6%) patients had a fresh embryo replacement accompanied by embryo cryopreservation, which was not significantly different from the EC group (62/182; 34.1%). The livebirth rate per fresh embryo transfer in the EC group (17/62; 27.4%) was significantly higher than that for the PN group (8/72; 11.1%; P < 0.05). Embryo survival following thawing was similar for the PN (96/129; 74.4%) and EC (79/102; 77.4%) stages. Although not significant, the livebirth rate following the transfer of thawed embryos was higher in the PN group (11/44; 25.0%) than in the EC group (4/38; 10.5%). Following one fresh and two freeze-thaw embryo replacements, the observed cumulative viable pregnancy rates were comparable for patients in both the PN (40.2%) and EC (41.1%) groups.      

Controlled ovarian hyperstimulation and intrauterine insemination for treating male subfertility: a controlled study

In this randomized crossover trial we investigated whether the use of controlled ovarian hyperstimulation with low-dose human menopausal gonadotrophin in couples with male subfertility leads to a higher probability of conception when intrauterine insemination (IUI) is applied. We also investigated whether the efficacy of IUI in natural or stimulated cycles was related to the severity of male subfertility. Seventy-four couples completed 308 treatment cycles. Thirteen pregnancies occurred after IUI in a natural cycle (pregnancy rate per completed cycle: 8.4%) and 21 after IUI in a stimulated cycle (pregnancy rate per completed cycle: 13.7%). The difference between the two treatment modalities was not statistically significant. The efficacy of IUI in stimulated cycles was related to the severity of the semen defect. In couples with a total motile sperm count < 10 x 10(6), ovarian stimulation did not improve treatment outcome, while it did in couples with a total motile sperm count > or = 10 x 10(6). Compared with the expected chance of conceiving spontaneously without treatment, both natural and stimulated cycles improved the probability of conception. We conclude that, for the group as a whole, ovarian stimulation did not improve the probability of conception. However, in couples with less severe semen defects, ovarian stimulation did improve the probability of conception.      

In vivo evaluation of signal processors for laser Doppler tissue flowmeters

The performance of different signal processors for laser Doppler tissue flowmeters was evaluated by the use of a well defined flow model comprising a segment of the feline intestinal wall. The processor that, apart from being based on the calculation of the first moment of the power spectral density, also takes into account the effect of multiple scattering in a number of blood cells gave an output signal that was linearly related to the intestinal wall perfusion as recorded independently by a drop-counting technique. At a recording bandwidth of 12 kHz, this linear relationship was valid for the entire flow range 0–300 ml min⁻¹ 100 g⁻¹ (r=0·98). The processor based on the first moment of the power spectral density alone under-estimated the highest flow rates by about 35 per cent, while within the flow range 0–100 ml min⁻¹ 100 g⁻¹ this processor also gave an output signal linearly related to flow at a recording bandwidth of 12 kHz (r=0·96). When the bandwidth was limited to 4 kHz, the output signals from both processors were linearly related to flow only within the range 0–100 ml min⁻¹ 100 g⁻¹ (r=0·90). The output signals recorded with the 4 kHz systems were, however, generally only about 65 per cent of those recorded with the 12 kHz systems.