Salmonella-specific monoclonal antibodies against recombinant Salmonella typhi 36-kilodalton porin.

Mouse monoclonal antibodies were raised against recombinant Salmonella typhi 36-kDa porin monomer. Specificities of 16 monoclonal antibodies were analyzed as reactivity patterns in dot immunobinding and Western blot (immunoblot) assays using isolated outer membrane proteins of gram-negative bacteria and cloned purified S. typhi porin monomers and trimers. Four monoclonal antibodies were specific for Salmonella spp.     

Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.     

Quantitative antimicrobial susceptibility test for Streptococcus pneumoniae using inoculum supplemented with whole defibrinated sheep blood.

The National Committee for Clinical Laboratory Standards recommends the use of lysed horse blood-supplemented Mueller-Hinton broth for determining the quantitative antimicrobial susceptibility of Streptococcus pneumoniae. This procedure may be difficult for laboratories using previously prepared or commercial MIC systems. Therefore, a study was undertaken to determine whether previously prepared microdilution trays containing Mueller-Hinton broth without blood could be used for determining the antimicrobial susceptibility of S. pneumoniae by adding whole defibrinated sheep blood to the bacterial suspension used to inoculate the trays. The presence of alpha-hemolysis was used as an indicator of bacterial growth. One hundred isolates of S. pneumoniae selected to represent a distribution of susceptibility patterns were tested by the National Committee for Clinical Laboratory Standards method and the sheep blood-supplemented-inoculum method. Greater than 94% agreement between the two methods was achieved. The sheep-blood-supplemented-inoculum procedure was highly reproducible and easy to perform and provides an acceptable alternative for determining the MICs for S. pneumoniae for laboratories using previously prepared or commercial microdilution systems.     

Altered regulation of mitogen responsiveness by suppressor cells in multiple sclerosis.

Suppression of the lymphocyte response to concanavalin A (Con A), phytohaemagglutinin (PHA) and protein A from Staphylococcus aureus (SpA) by Con A-induced suppressor cells was measured in twenty-four patients with recently active-recovering multiple sclerosis (MS), twelve with inactive MS and twenty-three healthy controls. Patients with recently active disease displayed significantly greater suppression of the response to Con A. Suppression of the responses to PHA and SpA did not differ among the groups. Lymphocyte stimulation in cultures not showing suppression was similar in all three types of subjects. These results suggest a disturbance of lymphocyte regulation in patients with recently active-recovering MS and illustrate the potential usefulness of measuring the suppression of responsiveness to several mitogens.     

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.      

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.