Citrate shows specific, dose-dependent lympholytic activity in neoplastic cell lines.

We have demonstrated cell membrane destruction activity by carboxylic acid derivatives (CADs) mainly tri-sodium citrate, in neoplastic cell lines and, to a far lesser extent, in normal human peripheral blood mononuclear cells (hPBMC). Flow cytometric (FACS) analysis was applied to Annexin-V and Propidium Iodide (PI) stained cells to evaluate the degree of the apoptosis induced by citrate in the following cell lines: CCRF-CEM (shortened to CEM), H9, and Jurkat (T-Cells), Raji and WIL2-NS (B-Cells), HL-60 (myeloblasts), K562 (myelocytes) and U937 (monocytes). We also tested normal hPBMC. Before staining with Annexin/PI, manual cell counts were performed on 24- and 48-h-old cell cultures. Cell supernatants were assayed for lactate dehydrogenase (LDH). LDH values in samples correlated with enhanced apoptosis by FACS analysis. For comparison, ascorbate and 2 other CADs including, acetate and lactate were also evaluated for the induction of apoptosis. In addition, the ability of tri-sodium citrate to induce apoptosis in the presence and the absence of several antineoplastic drugs, such as dexamethasone, arsenic trioxide, hydrocortisone, 6-mercaptopurine, and methotrexate were tested on Jurkat cells. FACS, LDH, and cell count values all demonstrated an enhanced degree of apoptotic cell death in Jurkat cells by citrate. In most of our investigated cells, except for the H9 cell line, citrate has induced a greater degree of apoptosis than acetate which induced a greater degree than lactate (see Fig. 1.0). The nature of the cell death by ascorbate appeared to be due to necrosis rather than apoptosis. Pilot studies on normal hPBMC showed that citrate alone or in combination with antineoplastic drugs caused minimal cell death. Thus citrate might be of benefit in some chemotherapy treatments in order to reduce drug toxicity or possibly enhance drug activities in certain neoplasias.

Bactericidal activity of citrate against Gram-positive cocci.

AIMS: Coagulase-negative staphylococci (CNS) are now important nosocomial pathogens and are usually resistant to multiple antibiotics. Citrate is an alternative antimicrobial product which has been used as a preservative. METHODS AND RESULTS: In this pilot study the bactericidal activity of Na citrate against 10 isolates of CNS and 6 other gram-positive pathogens was examined and compared with that of Na lactate. CONCLUSIONS: All staphylococci tested were susceptible (> or = 2 log(10) killing) to citrate at 6.25-25 mg ml(-1). Na lactate showed only modest killing at 50 or 100 mg ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: Citrate may warrant broader evaluation as an antimicrobial additive such as in topical agents.

Nasal colonization by methicillin-resistant coagulase-negative staphylococcus in community skilled nursing facility patients.

BACKGROUND: Methicillin-resistant coagulase-negative staphylococci (MRCNS) are increasing nosocomial pathogens in acute care hospital patients. However, there is little information on the epidemiology of MRCNS in skilled nursing facilities (SNFs). We report a pilot survey of the prevalence of MRCNS colonization in SNF patients. METHODS: Anterior nasal swabs were plated on oxacillin salt screening agar for selection of MRCNS. Suspected MRCNS were confirmed by coagulase and catalase tests and standard disc-diffusion antimicrobial susceptibility tests. RESULTS: The overall prevalence of MRCNS was 40% for in-house continuing SNF patients, 49% for newly admitted patients, and 60% for SNF nursing personnel. The prevalence was 13% in a "control" group of nonmedical personnel. Forty-six percent of MRCNS were resistant to ciprofloxacin. The frequency of colonization with MRCNS increased over time. After an average 17 months of facility stay, 32% of noncarriers acquired MRCNS. High frequency of colonization was associated with greater disability. CONCLUSION: Colonization with MRCNS is common among SNF patients, who can serve as a reservoir for transfer of such strains to acute care hospitals. Careful infection control practice, including judicious use of antibiotics with frequent handwashing, will remain critical policies for limiting spread of such strains.

Nasal colonization by Staphylococcus aureus in active, independent, community seniors.

OBJECTIVE: to evaluate the prevalence of nasal colonization with Staphylococcus aureus (SA) in active, independent community seniors and old people in a nursing home. DESIGN: cross-sectional brief questionnaire and screening culture of anterior nares specimens from 165 elders at a community centre and cross-sectional data from a recent survey in a nursing home. RESULTS: the prevalence of SA colonization in community seniors (27%) was similar to that in the nursing home (29%). The proportion of SA isolates that were methicillin-resistant was much lower in the community seniors (2.3%) than in the nursing-home residents (31%). There was less antibiotic resistance in those living at home. CONCLUSION: in community seniors the prevalence of SA colonization was similar to that in nursing-home residents, but the prevalence of methicillin-resistant SA was lower. Susceptibility patterns of antibiotics tested against the SA showed less resistance than isolates from nursing-home patients.

Low-level colonization and infection with ciprofloxacin-resistant gram-negative bacilli in a skilled nursing facility.

BACKGROUND: We report a 1-year surveillance study that evaluates colonization and infection with ciprofloxacin-resistant gram-negative bacilli (CR GNB) and the relation to quinolone use and other possible risk factors in a proprietary skilled nursing facility (SNF) with no history of outbreaks. METHODS: Rectal swabs obtained quarterly were streaked on MacConkey agar with ciprofloxacin discs (5 microg) to screen for CR GNB and later were speciated and the antimicrobial susceptibilities were confirmed by standardized disc-diffusion tests. RESULTS: The mean prevalence of CR GNB colonization was 2.6% (range 0.9% to 5.3%). The colonization frequency was higher in the last survey than it was in the first survey. CR GNB-colonized strains included Pseudomonas species (21%), but more than half were non-Pseudomonas enterics such as Acinetobacter baumannii (25%), Proteus mirabilis (17%), and Providencia stuartii (13%). None of the patients who had colonization with CR GNB had subsequent infections with the same species. Patients with colonization had more exposure to ciprofloxacin and they were more likely to have been recently admitted from an acute-care hospital and have decubitus ulcers. During the study period, of 336 patients surveyed, 98 (29%) patients developed suspected infections and cultures were done; the infection rate was 4.7 per 1000 patient days. Of these infected patients, 59 (60%) were infected by GNBs; the infection rate was 2.3 per 1000 patient days. Nineteen percent of the GNB infections were treated with a quinolone. (Overall, quinolones constituted about 17% of antibiotic usage in the SNF). Only 3 (5%) of the patients infected with GNB were infected with CR GNB, including Pseudomonas and Providenci a species. The CR GNB infections involved multiple sites, multiple organisms, and long length of stay in the SNF. CONCLUSIONS: The findings indicate that in this community SNF, a low frequency of colonization or infection with CR GNB existed. Whether continued moderate use of quinolones will lead to increasing levels of CR GNB will require further study.

The effect of calcium-related factors on the predominance of IFN-gamma or interleukin-4 in cultured mononuclear cells.

Certain factors have been studied that might influence whether interferon-gamma (IFN-gamma) or interleukin-4 (IL-4) will predominate in cultures of peripheral blood mononuclear cells (PBMC). ELISA was used to measure cytokine protein, and PCR was used to quantitate mRNA. It was found that induction with plant lectins produced greater yields of IFN-gamma than induction with ionophores, but ionophores alone produced at least equal yields of IL-4 as did lectins. Phorbol myristate acetate (PMA) greatly enhanced IFN yields in the presence of ionophores but had no significant influence on IL-4 production. Calcium-independent cytokine induction using anti-CD28 and PMA resulted in production of both cytokines, whereas depletion of extracellular calcium and magnesium adversely influenced the yield of both IL-4 and IFN-gamma. Finally, calmidazolium, an inhibitor of calmodulin, had an enhancing effect on IFN-gamma yields when phytohemagglutinin (PHA) was the inducer and an adverse effect when A23187 was the inducer. In contrast, calmidazolium reduced IL-4 yields with both PHA and A23187 induction.

Surveillance of colonization and infection with Staphylococcus aureus susceptible or resistant to methicillin in a community skilled-nursing facility.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen in acute care hospitals and long-term care facilities. Few studies have been reported in private skilled nursing facilities (SNFs) not experiencing outbreaks of infections caused by MRSA. METHODS: From a 149-bed SNF with no outbreaks, we report a 1-year prospective surveillance study of S. aureus colonization and infection, with focus on S. aureus phenotypes, both methicillin susceptible (MS) and methicillin resistant (MR). Nasal and stool or rectal screening cultures were done on admission, and all patients underwent screening on at least a quarterly basis for 1 year. RESULTS: Overall, 35% of patients were colonized at least once with S. aureus, (72% MS, 25% MR, and 3% mixed phenotypes), 94% of the MRSA were ciprofloxacin resistant. Nasal colonization with any S. aureus was more frequent, but 13% of patients had positive results only in rectal specimens. Twenty-one percent of the newly admitted and 15% of continuing patients acquired colonization during their stay in the SNE Colonization was transient or persistent, persisted longer in the nares compared with colonization in rectal specimens, and was more stable for methicillin-susceptible S. aureus. Nine percent of patients had development of infection with S. aureus. There was no indication that MRSA colonization led to more infections than methicillin-susceptible S. aureus. Of the 13 infected patients in whom cultures had previously been obtained, seven (54%) had been colonized by the same phenotype strains. CONCLUSIONS: In this private SNF, endemic S. aureus infections occur at a low frequency, reflecting a moderate level of colonization with S. aureus. However, a trend showing gradual increases in frequencies of colonization and infection is of concern and suggests that in this SNF, future intervention could become warranted.

Platelet-activating factor and the production of human interferon-gamma.

Platelet-activating factor (PAF) was tested for its ability to alter yields of human interferon (IFN) produced from peripheral blood mononuclear cells (PBMC). Using different concentrations of phytohemagglutinin (PHA) we could not demonstrate a consistent effect of PAF at any concentration tested on the yield of IFN-gamma. Similarly, although PAF was associated with a slight enhancement of IFN-gamma yields when PBMC were induced by interleukin-2 (IL-2), the results were not statistically significant. No effect was observed on the accumulation of human IFN-gamma mRNA induced by PHA. Furthermore, PAF did not enhance yields of IFN-gamma when the cells were induced by poly I:poly C. We conclude that although PAF may have a role in sepsis, it is not likely that this is in any way related to its ability to significantly alter the yield of interferons.

Colonization by Staphylococcus aureus resistant to methicillin and ciprofloxacin during 20 months' surveillance in a private skilled nursing facility.

OBJECTIVE: To evaluate endemic colonization with Staphylococcus aureus resistant to methicillin, ciprofloxacin, or both among patients of a private skilled nursing facility, with regard to colonization rate and site, and relation to infection and prior antibiotic use. DESIGN: Prospective quarterly culture surveillance of nares and rectal specimens over 20 months' observation. RESULTS: The mean prevalence was 3.8% in new admissions and 5.4% for in-house patients; cumulatively, 7.5% of the patients were colonized during the study period. The colonization rate remained stable during the study period. Screening of rectal, as well as nares, specimens detected substantially more colonized patients than would have been detected by nasal cultures alone. Five to seven percent of the colonized patients developed later infection with methicillin-ciprofloxacin-resistant S aureus. Colonized patients did not differ significantly from the noncolonized group in prior use of quinolones, but the colonized group was exposed significantly more frequently to other antibiotics than the noncolonized group. Eighty-three percent of methicillin-resistant S aureus (MRSA) isolated from infections and 89% from colonization were also ciprofloxacin resistant. CONCLUSION: Although all infecting and most colonizing isolates of MRSA were resistant to quinolones, the overall rate of colonization remained low and stable despite the continued use of quinolones. The findings suggest that good infection control practice has prevented broader spread of such strains in this facility.

Infection surveillance and antibiotic utilization in a community-based skilled nursing facility.

To survey the types of suspect infections, the antibiotic utilization and the patterns of antibiotic resistance among bacterial pathogens in a community Skilled Nursing Facility (SNF), we conducted a 20-month prospective observational surveillance program comprising all 585 patients admitted to a 149-bed private community SNF. Data were collected form medical charts, laboratory reports and nurses reports. Overall, 41% of the patients developed at least one presumptive nosocomial infection, and 54% of the patients received one or more antibiotic treatments. The overall presumptive nosocomial infection rate was 7.2 per 1000 patient days. The most common sites of presumptive nosocomial infection were the urinary tract (38%) and the respiratory tract (28%). The most common pathogens overall were E. coli (25%). Antibiotic groups used most frequently were the quinolones (22% of prescriptions). Thirty-nine percent of the Staphylococcus aureus isolates associated with suspected infections were resistant to methicillin, and of these 94% were also resistant to ciprofloxacin. Most of the resistant S. aureus isolates were from indwelling catheter-associated with UTIs. Infections associated with quinolone resistant strains of gram-negative bacilli were infrequent. No epidemiologic evidence of nosocomial clustering was apparent.

Effect of hemodialysis on leukocyte adhesion receptor expression.

Hemodialysis with complement-activating membranes such as cuprophane is known to transiently activate leukocytes, leading to increased cellular adhesiveness, pulmonary leukostasis, and reduced functional capacity of monocytes and neutrophils. Clinically, this repetitive cell activation may contribute to the increased morbidity and mortality associated with chronic hemodialysis. To examine the effect of cuprophane hemodialysis on expression of cell-surface proteins involved in leukocyte adhesiveness, we monitored CD11b, CD18, CD14, CD54, and plasma-soluble CD54 in 10 patients during hemodialysis with cuprophan dialyzers. To test the effect of local blood recirculation, in two patients, arterial supply to the dialyzer was accessed from the peripheral arteriovenous fistula and was returned via an indwelling central venous catheter. In an attempt to examine the possible role of membrane-induced complement activation, the results were compared with those seen after incubation with C5a in vitro. Finally, the leukocyte responses to C5a and lipopolysaccharide were measured before and after hemodialysis. Leukocyte expression of CD11b and CD18 increased and CD14 decreased with hemodialysis, while CD54 remained unaltered. Plasma CD54 was markedly elevated before and remained unchanged during hemodialysis. Data obtained with C5a activation in vitro revealed identical changes in CD11b expression as that seen with hemodialysis, suggesting the role of membrane-induced complement activation. Preliminary data obtained using remote arterial and venous access sites showed only a slight increase in CD11b expression in the arterial blood, suggesting that the apparent systemic activation seen with arteriovenous access may be due to recirculation and local activation within the blood access. Finally, dialysis procedure did not impair lipopolysaccharide- or C5a-mediated upregulation of CD11b expression.

Bacteremia due to Haemophilus infections: a retrospective study with emphasis on the elderly.

We performed a retrospective study of all patients in a large health maintenance organization in Southern California who were identified as having positive blood cultures for Haemophilus organisms during a 20-month period (September 1990 to May 1992) to assess the incidence, presentation, and predisposing conditions of bacteremia due to these organisms and to examine some of the features of these infections in the elderly. Thirty-eight patients with bacteremia due to haemophilus infections were identified. Ten (26.3%) patients were 65 years of age or older. The incidence of bacteremic haemophilus infections in the elderly group was estimated at 2.7 per 100,000 individuals per year, which was almost three times greater than that for the younger age groups studied. When analyzed statistically, the presenting feature of the infection did not differ among age groups. Six patients died, four of whom were elderly. All six deaths were due to nontypable Haemophilus influenzae strains. Cancer was the only chronic underlying condition frequently found among the elderly patients. Three of 10 elderly patients lived in nursing homes; all three were infected with nontypable H. influenzae strains, and all three died.

Flow cytometric investigation of neutrophil activation pathways by n-formyl-Met-Leu-Phe and phorbol myristate acetate.

Recent evidence suggests that multiple pathways exist in PMN activation and that specific leukocyte response may be due to the activation of a particular signaling pathway. Using flow cytometry, PMN activation pathways were studied through the parallel comparison of n-formyl-Met-Leu-Phe (fMLP)- and phorbol-12-myristate-13-acetate (PMA)-induced stimulation and by simultaneous assays for CD11b expression and morphology. The maximal CD11b expression was higher with PMA than with fMLP, suggesting different activation pathways. Under these experimental conditions, a morphological response to fMLP was not observed. However, significant shape change was detected in PMA treated samples and was suppressed by either the removal of extracellular calcium or staurosporine at the concentrations above 14.5 microM. Calcium ionophore induced a similar light scattering pattern to that by PMA and enhanced CD11b expression, both of which were not inhibitable by staurosporine. These observations, for the first time, indicated that Ca2+ was a mediator in activation processes and that the treatment of PMN with PMA resulted in Ca2+ influx.

Colonization by Staphylococcus species resistant to methicillin or quinolone on hands of medical personnel in a skilled-nursing facility.

We report here a pilot survey of colonization with methicillin- and/or ciprofloxacin-resistant Staphylococcus species on hands of nursing personnel in a private skilled-nursing facility. We found only one nurses aide who carried methicillin-resistant Staphylococcus aureus and one who carried ciprofloxacin-resistant S. aureus, each on only one of the surveys. None of the control nonmedical personnel were found to carry methicillin-resistant S. aureus or ciprofloxacin-resistant S. aureus. The colonization rate of methicillin-resistant coagulase-negative staphylococci on the hands of medical personnel was 59%, compared with 13% for the nonmedical personnel, and the counts of methicillin-resistant coagulase-negative staphylococci were also significantly higher for nursing personnel. For ciprofloxacin-resistant coagulase-negative staphylococci, 30% of nursing personnel had positive cultures whereas no ciprofloxacin-resistant coagulase-negative staphylococci strains were recovered from the nonmedical control cohort. Three of the patients had presumptive infections with methicillin- or ciprofloxacin-resistant coagulase-negative staphylococci, all urinary tract infections. Personnel hands represent a likely mode of transmission of such strains between patients, and skilled-nursing facility patients may represent a reservoir for carrying the coagulase-negative staphylococci back to acute care facilities.

Induction, transcription, synthesis, and adsorption of interleukin-1 by dialyzer membranes.

This study was designed to dissect the direct effect of dialyzer membrane on interleukin-1 (IL-1) induction from those of complement activation, mechanical stimulation, acetate/bicarbonate and endotoxin diffusion, and cell type interactions. To this end, a suspension of P388D1 murine macrophages in a complement-free culture medium containing 10% heat-inactivated serum, a closed-loop system consisting of tubing alone or with a hollow-fiber cuprammonium cellulose (CU) or polyacrylonitrile (PAN) dialyzer, and a roller pump were used. The dialysate compartment was filled with the same medium and capped. Cell suspension was recirculated at 300 mL/min for 3 h. Cells and supernates were separated, and adhering proteins were eluted. All samples tested negative for endotoxin. IL-1 mRNA was greatest with CU, followed by PAN and tubing alone. IL-1 in the supernate was greater with CU than with either tubing alone or PAN (P < 0.005; analysis of variance), which showed comparable values. IL-1 eluted from loops was greatest with PAN dialyzers, followed by sets with CU dialyzers and tubing alone (P < 0.001; analysis of variance). Thus, both CU and PAN membranes directly induce IL-1. However, avid adsorption by PAN attenuates the rise in circulating IL-1.

Enhanced yields of gamma interferon in prolactin treated human peripheral blood mononuclear cells.

Prolactin is a peptide hormone with effects on a number of target organs including the immune system. It has been shown that animals rendered hypoprolactinemic have impaired delayed hypersensitivity, impaired macrophage activation and altered secretion of gamma interferon (IFN). Using peripheral blood mononuclear cells (PBMC) and inducing the cells to produce gamma IFN with a range of inducers, we have studied the effects of a number of hormones on IFN production. Using cells from normal donors, we have found that prolactin in concentrations of 10(-8) M or greater, can significantly enhance the production of gamma IFN. The effect was dose related and was observed when lectins (PHA and Con A), but not anti CD3 antibodies, ionophones, or IL-2 were used to induce the cells. The presence of prolactin in concentrations above that encountered in the fetal bovine serum used to incubate the cells resulted in a doubling or more of the IFN produced. The tests were performed on 30 occasions with cells drawn from 21 individuals. On all but three occasions, yield enhancement was observed in the presence of prolactin. The mechanism of the effect was investigated, and genistein, a tyrosine kinase inhibitor, was found to abort the influence of prolactin on gamma IFN production. These studies indicate prolactin in physiological concentrations can enhance the production of gamma IFN from cells from normal donors.

The effect of gonadotropins on the production of human interferon-gamma by mononuclear cells.

Gonadotropins--follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (HCG)--and the related agent thyrotropin were shown to enhance yields of interferon-gamma (IFN-gamma) from human peripheral blood mononuclear cells (PBMC) significantly when calcium ionophores (ionomycin or A23187) were used as inducers. The enhancement increased the IFN yields four- to eight-fold. Induction with other inducers, (such as lectins, interleukin-2 (IL-2), and anti CD3, was not associated with enhancement of the IFN yields by gonadotropins. Concentrations of gonadotropins associated with pregnancy (HCG) or menopause (FSH and LH) were able to enhance IFN-gamma yields. Addition of the gonadotropins to the cells after the ionophore gave the greatest degree of enhancement. Perturbation of the calcium messenger system or nonspecific stimulation of adenyl cyclase failed to influence the IFN yield enhancing effect of the gonadotropins. No effect of gonadotropins was observed on IFN bioactivity.