On-probe sample pretreatment for the detection of proteins above 15 KDa from whole cell bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A rapid methodology is described for the enhancement of the signal-to-base-line (S/B) ratio of high molecular weight protein signals from whole cell bacteria analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The procedure involves depositing growing bacteria colonies from culture dishes directly onto the MALDI probe followed by treatment of the sample spot with a 2 microL aliquot of 40% ethanol prior to the addition of a ferulic acid matrix solution (12.5 mg dissolved in 17% formic acid/33% acetonitrile/50% H(2)O). Protein signals of more than 20 kDa were routinely produced from both Gram positive and Gram negative bacteria prepared in this manner. Moreover, a substantial number of intense protein signals were also produced in the more 'conventional' fingerprint region extending from 4 to 20 kDa. This approach is rapid, easy to implement into existing methodologies, and does not require any special hardware.

Holy water--a risk factor for hospital-acquired infection.

A case of hospital-acquired infection due to Acinetobacter baumanii in a burns patient after exposure to holy water is described. In order to assess the infection risk, 13 samples of holy water were cultured for bacteria, (including legionellae) and yeasts. Viable bacterial counts ranged from 1 center dot 3 x 10(3)-3 center dot 8 x 10(8) cfu/L (mean 3 center dot 1 x 10(7) cfu/L). A wide range of bacterial species was isolated including Pseudomonas aeruginosa, Enterobacter spp. Escherichia coli and Aeromonas hydrophila. Candida spp. were isolated from two samples, but legionellae from none. Holy water would, therefore, seem to be a potential risk factor for hospital-acquired infection.

Changes in lymphocyte subset distribution aid in the differential diagnosis of renal allograft dysfunction.

The distribution of selected lymphocyte subset populations in renal transplant patients was used to assist in the differential diagnosis of graft dysfunction. Patients experiencing dysfunction due to rejection showed consistent and significant decreases in relative numbers of CD 8 (cytotoxic/suppressor) lymphocytes. The ratio of CD 4 cells to CD 8 cells in this group of patients was generally greater than 2.00 due to decreases in CD 8 cells. Patients showing graft dysfunction due to viral infections showed consistent and significant increases in CD 8 cells which also bear the HNK-1 or the HLA-Dr determinants. Serial monitoring for these dual-marked lymphocytes on a weekly basis can be of considerable use in determining the etiology of graft dysfunction. Increases in other "activation" markers, including transferrin receptors, CD 38, and a T cell lineage specific activation antigen (TLiSA) were not specific for rejection; in fact, increases in CD38 were more often associated with viral infections. These studies indicated that lymphocyte subset determinations done on a regular basis can help distinguish graft dysfunction due to viral infections from other causes. The ability to distinguish rejection episodes from stable grafts is less obvious. Although the alterations in lymphocyte subset distribution are not entirely specific, they can distinguish viral infections from rejection.

Effect of sublethal gamma radiation on host defenses in experimental scrub typhus.

The effect of sublethal gamma radiation on inbred mice chronically infected with scrub typhus rickettsiae was examined. Inbred mice which were inoculated with the Gilliam or Karp strain of Rickettsia tsutsugamushi by the subcutaneous route harbored the infection for at least 1 year. Irradiation of these animals at 12 or 52 weeks postinoculation with normally sublethal levels induced a significantly higher percentage of rickettsemic mice (recrudescence) than was seen in the unirradiated, similarly infected control animals. In addition, sublethal irradiation at 12 weeks induced a quantitative increase in total rickettsiae. Homologous antibody titers to the rickettsiae were examined for 5 weeks after irradiation to determine the role of the humoral response in radiation-induced recrudescence. Unirradiated, infected mice showed consistent titers of about 320 throughout the 5-week observation period, and the titer was not affected by exposure of up to 500 rads of gamma radiation. Drug dose-dependent radioprotection and modification of recrudescence was noted in infected, irradiated mice treated with the antiradiation compound S-2-(3-aminopropylamino)ethyl phosphorothioic acid. The results of this investigation supported the conclusion that the recrudescence of a chronic rickettsial infection in the appropriate host after immunological impairment due to gamma radiation can result in an acute, possibly lethal rickettsemia.

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate.

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.

Release of soluble "blocking" and "suppressor" factors from normal lymphocytes treated with RNA from spleens of tumour-bearing mice.

RNA extracted from the spleens of tumour-bearing (TLRNA) and tumour-immune (ILRNA) mice was shown to transfer to normal lymphocytes (NL) the ability to produce factors that blocked specific tumour-cell cytotoxicity and mediated specific antibody-dependent cell cytotoxicity (ADCC). Aliquots of normal C3H mouse lymphocytes were treated with TLRNA or ILRNA and cultured in vitro in the absence of tumour antigen. Supernatants were collected at 24h intervals and tested in a microcytotoxicity assay for blocking and ADCC activities. Factors that inhibited tumour destruction by specifically sensitized lymphocytes at the level of both the tumour cells and effector cells were demonstrable in culture supernatants of NL pretreated with TLRNA (50 or 100 microgram/4 X 10(6) cells) but not ILRNA. However, treatment of NL with either RNA resulted in the production factors that mediated tumour-specific ADCC. Cytotoxicity testing and absorption studies of the tumour cell and a control cell (LM) indicated that factors mediating ADCC and blocking at the target-cell level were specific for the tumour. Suppressor activity at the effector-cell level was not absorbed by tumour cells and represents a separate and distinct mechanism of immunosuppression. These data indicate that RNA faithfully transfers "suppressive" as well as "positive" types of immune responses that have been reported previously for lymphocytes obtained directly from tumour-bearing and tumour-immune animals.

Cellular immunity in neoplasia. Antigen and mitogen responses in patients with bronchiogenic carcinoma.

Cellular immune responses of patients with histologically confirmed lung carcinoma were assessed in vivo using cutaneous response and in vitro with a microlymphocyte blastogenic transformation (LBT) assay. In addition, correlation of the cutaneous response with the migration inhibitory factor (MIF) assay and LBT response was examined. The results indicated that cutaneous responses seen in patients with cancer of the lung were consistently lower than similar responses in normal controls (p less than 0.001). Similarily, the percentage of positive cutaneous responses seen with patients included in this study was lower than the frequencies reported by others. Stimulation of cells from lung cancer patients by PHA-M was also depressed when compared to similar lymphocytic responses in normal volunteers (p less than 0.001). The correlation between cutaneous response to tuberculin and the in vitro assays was high. The few instances of disparity demonstrate the need to utilize more than one assay in evaluating cellular immune functions. These data would support the work of others that indicate a depression of cellular immunity in advanced malignancy.