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.

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.

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.

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.

Viral infections.

Viral infections may cause serious morbidity as well as death in elderly patients. Of the RNA viruses, influenza virus is the most important pathogen; the majority of influenza-related deaths occur in older patients. Respiratory syncytial virus appears to be gaining increasing importance in elderly persons. Herpes zoster or shingles is caused by the DNA virus, varicella-zoster virus, and its major morbidity in older patients is postherpetic neuralgia.

The paradoxical effects of somatostatin on the bioactivity and production of cytotoxins derived from human peripheral blood mononuclear cells.

Somatostatin (SMS), a naturally occurring peptide is known to inhibit the production of certain protein molecules and to diminish the ability of peripheral blood mononuclear cells to proliferate. We tested the effects of three forms of SMS on the bioactivity of both lymphotoxin (LT) and tumour necrosis factor (TNF). We also tested the effects of these agents on production of cytotoxins by peripheral blood mononuclear cells. We found the 28 amino acid form of SMS significantly enhanced the bioactivity of both LT and TNF (10(-9) M concentration) when tested in mouse L cells. The 14 amino acid form of SMS enhanced LT (10(-9) M concentration) activity but not TNF activity. The first 14 amino acid form of SMS-28 (amino terminal) did not affect bioactivity of the cytotoxin. In contrast, the naturally occurring 14 amino acid form of SMS (10(-8) M concentration) significantly diminished production of cytotoxin by human peripheral blood mononuclear cells. Cytotoxin produced by the latter was shown to be a combination of both LT and TNF. Similarly after SMS exposure, the cytotoxin produced remained a mixture of LT and TNF in roughly similar proportions. It thus appears that certain forms of SMS can enhance the bioactivity of cytotoxins, but at the same time decrease the production of these cytotoxins.

Functional, biochemical, and histopathologic consequences of high-dose interleukin-2 administration in rats.

A variety of side effects have been reported with the use of interleukin-2 alone or in combination with lymphokine-activated killer cells in patients with disseminated neoplasms. The present study was undertaken to determine the effects of high-dose interleukin-2 administration in normal rats. Sprague-Dawley rats were treated with intravenous recombinant interleukin-2 (900,000 IU/kg/day) for 9 consecutive days. Animals were placed in individual metabolic cages, and arterial blood pressure, food intake, body weight, and urine output were monitored. On day 10, animals were killed by exsanguination, various tissues were harvested, and a variety of hematologic and chemical assays were performed. The results were compared with those of placebo-injected normal control and pair-fed groups. The interleukin-2-treated group exhibited anorexia, weight loss, hypotension, anemia, leukocytosis, lymphocytosis, eosinophilia, hypercalcemia, azotemia, and a marked urinary concentration defect. Histologic examination of various tissues revealed widespread infiltration with mono-nuclear cells and eosinophils in most organs, especially in the lungs and liver of interleukin-2-treated animals. Other abnormalities included severe panlobular hepatitis, hepatocellular necrosis, and thymic involution. Renal involvement was mild and consisted of focal interstitial infiltration by mononuclear cells. According to these observations, administration of high-dose interleukin-2 in normal rats results in a score of significant functional, biochemical, and histologic abnormalities.

Cytokine levels during hemodialysis.

The authors measured plasma IL-1 (interleukin-1), IL-2, IL-2 receptor (IL-2R), IL-4, IL-6, tumor necrosis factor (TNF), and granulocyte macrophage colony stimulating factor (GMCSF) in 10 stable patients during hemodialysis (HD) using new or reused polyacrylonitrile (PAN) or cuprophan (CU) dialyzers. Five HD patients with wasting syndrome, and 16 normal controls, were included. Hemodialysis patients showed a marked elevation of IL-2R. No IL-4, IL-6, or GMCSF were detected in any group. Tumor necrosis factor and IL-2 in the HD group were comparable with control values. No difference was found in the TNF levels in HD patients with and without wasting syndrome. Cytokine levels were unaffected by either new or used PAN or CU dialyzers.

The effect of vitamin D3 on the production of cytotoxic factors by human peripheral blood mononuclear cells.

Vitamin D3 at a concentration of 300 nM was shown to significantly reduce the yields of cytotoxin produced by human peripheral blood mononuclear cells. Reduction of cytotoxin titers occurred when induction was performed with either concanavalin A, interleukin 2, or ionomycin. Typing of this cytotoxin was accomplished using antisera that were prepared by immunization of rabbits with either recombinant tumor necrosis factor or lymphotoxin. Both antisera were demonstrated to neutralize the cytotoxic activity produced by the peripheral blood mononuclear cells in the presence or absence of vitamin D3. These latter experiments suggested that the cytotoxin in question might be lymphotoxin. Vitamin D3 was also shown to decrease cytotoxin production by a transformed B-cell line known to produce only lymphotoxin, supporting the concept that this vitamin could diminish yields of lymphotoxin at least under certain circumstances. However, purified adherent cells induced by Escherichia coli lipopolysaccharide produced a cytotoxin that was also inhibited by the presence of vitamin D3. Indomethacin failed to influence the effects of vitamin D3 on the production of the cytotoxin. These studies suggest that vitamin D3 may have a role in regulating the secretion of cytotoxic factors which may be important in the defense against neoplastic diseases.

The regulation of interferon production by aspirin, other inhibitors of the cyclooxygenase pathway and agents influencing calcium channel flux.

Interferon is a family of potent antiviral agents which can activate macrophages, enhance cell surface markers, or influence antibody production. Three major types of human interferon are known to exist and have been designated interferons alpha, beta, and gamma. Because of its unique antiviral properties and its ability to influence the immune response, interferon has long been considered a potential therapeutic intervention in the treatment of infections and possibly neoplastic diseases. Two potential means to utilize interferon might be considered: One method would involve the administration of exogenous interferon, but an alternative might augment natural interferon production. We have been investigating a series of pharmacological agents that might influence its production and action. Since prostaglandins influence the immune response, we have investigated the effect of these cyclic fatty acids and those agents that influence their production on soluble protein mediators of the immune response on interferon. Our studies have focused on the effects of acetylsalicylic acid on the interferon system. We have demonstrated that prostaglandins of the E series can significantly reduce the yields of human interferon gamma, but not alpha (the two species of leukocyte derived interferon). In general, yields of gamma interferon produced by peripheral blood mononuclear cells in the presence of PGE2 (0.1 to 0.01 ugm/ml) were approximately 15% of those produced in the absence of these substances. In contrast, when acetylsalicylic acid (10 ugm/ml) was added to the cultures of peripheral blood mononuclear cells yields of gamma interferon increased more than threefold. When examining the effects of acetylsalicylic acid on human alpha interferon production, we were also to enhance interferon harvests although we could not demonstrate an adverse effect of prostaglandins on the production of these bioactive proteins. Addition of acetylsalicylic acid and prostaglandins simultaneously to our cultures had a negative effect on gamma interferon production, but still was associated with enhanced yields of interferon alpha. In examining how prostaglandins might influence interferon production, we began to study other cellular requirements for lymphokine production including those processes which were calcium dependent. Preliminary studies demonstrated that production of human interferon gamma by peripheral blood mononuclear cells was calcium dependent, but production of human interferon alpha was not. Thus, almost all agents studied that influenced calcium dependent intracellular processes influenced the titer of human interferon gamma produced, but not that of human interferon alpha. In examining this phenomenon more closely we noted the calcium channel flux was critical to the production of interferon gamma, hence agents enhancing channel flux(Bay K 8644) increased the production of human interferon gamma, but agents diminishing channel flux (specifically) channel blockers diminished production of this interferon species. These effects depended on the nature of the specific inducing agent. We are now examining the relationship of our observations with calcium to our earlier work with acetylsalicylic acid.

The effect of beta-estradiol, progesterone and testosterone on the production of human leukocyte derived interferons.

Sex hormones including estrogens, progesterone and testosterones are known to have adverse effects on the immune system and particularly on the proliferative response. Since cytokine production is known to be dissociable from the proliferation of lymphocytes and since other steroid hormones profoundly affect cytokine production, we felt it would be important to know the effect of sex steroids on the production of interferons (IFN), particularly since the latter are known to be key substances in the immune response. We have shown estradiol can slightly reduce gamma IFN yields with certain inducers (Con A, SEA) but only in pharmacologic concentrations. Similarly, progesterone had a modest effect in the same concentrations but only when Con A was the inducer. Testosterone did not effect IFN titers at any concentration. None of the sex steroids affected alpha IFN production and none of them influenced the bioactivity of either IFN species. In all cases these hormones diminished proliferative responses as has been previously noted.

Mycobacterium avium complex infection presenting as endobronchial lesions in immunosuppressed patients.

Infections caused by Mycobacterium avium in relatively immunocompetent hosts usually occur with isolated pulmonary involvement. Patients with the acquired immunodeficiency syndrome (AIDS) more commonly have disseminated disease. Endobronchial masses, however, have not been described with M. avium complex infections in normal or compromised hosts. We describe the cases of two patients with AIDS, both receiving zidovudine therapy, who presented with endobronchial obstruction and hilar adenopathy. Results of radiographic and endobronchial examination of these lesions suggested malignancy. Histologic and microbiologic tests, however, showed M. avium, and the lesions responded remarkably to mechanical debridement and antimycobacterial therapy.

Changes in circulating monocytes in patients with progressive systemic sclerosis.

Circulating monocytes in 30 patients with progressive systemic sclerosis (PSS, scleroderma) and 28 age and sex matched normal controls were studied. Binding of the lectin peanut agglutinin (PA) was significantly reduced in PSS monocytes (p less than 0.001) together with a reduction in the density of nonspecific esterase staining (p less than 0.001) suggesting advanced maturation. Using monoclonal antibodies to identify cell surface markers, we demonstrated a significant reduction in PSS monocytes bearing the Leu M2 antigen (Mac 120, antigen presenting cells) over controls (p less than 0.05), but were unable to show any differences in the monocyte subpopulations using antisera against Leu M3 and HLA-DR surface antigens. The ectoenzymes 5'-nucleotidase (5'N) and alkaline phosphodiesterase 1 (APD1) were lower and leucine aminopeptidase (LAP) levels were higher in patients with PSS, compatible with immune activation. Interferon-gamma levels in serum did not appear to account for these changes, whereas the levels of Clq binding complexes correlated inversely with the levels of LAP (p less than 0.05). There was a strong correlation between the number of Leu M3 positive cells and the level of the ectoenzyme LAP (p less than 0.001). With increasing disease duration, higher levels of Clq binding complexes were detected (p less than 0.05). These results indicate that monocytes in PSS differ from those in normals and appear to have undergone advanced differentiation and activation changes.

Evaluation of the in vitro production of interferon gamma and other lymphokines in uremic patients.

It has been suggested that a deficient immune response can be responsible at least partially for the high risk of infections and neoplasia in uremic patients. Since interferon (IFN) is critical to the immune response, we have evaluated the in vitro production of IFN-gamma and other lymphokines by peripheral blood mononuclear cells (PBMC) drawn from patients with end-stage renal disease and appropriate controls. We have correlated production of lymphokines by these cells with proliferative response to different mitogens. It was found that the secretion of IFN-gamma in response to all three mitogens was elevated in these patients compared with the control group. This elevation was significant with both phytohemagglutin and staphylococcal enterotoxin A, but not with Con A. No significant difference was observed in production of lymphotoxins, IL-2, and leukocyte migration inhibition responses. In contrast the proliferative response appeared diminished in the PBMC of uremic patients. We concluded that defective lymphokine generation is not a major immunological problem in patients with end-stage renal disease. Indeed, they appear to release excess amount of IFN-gamma which is known to be a macrophage-activating factor. It is suggested that high IFN-gamma activity could enhance the secretion of IL-1 or endogenous pyrogen and result in development of febrile reactions in dialysis patients.

The effect of corticosteroids and other antineoplastic agents on the generation of leukocyte migration inhibition factor.

We have shown that hydrocortisone in physiological concentrations can inhibit the production of leukocyte migration inhibition factor (LMIF), but does not diminish the action of this lymphokine. Other agents tested failed to influence LMIF production. Inhibition of LMIF production by corticosteroids was influenced by the nature of the stimulus used for the production as an effect could be seen with PHA or Con A, but not Staphylococcal enterotoxin A (SEA). Production of LMIF was promptly restored after removal of the steroids. Furthermore, addition of a calcium ionophore to PHA restored the production of LMIF even in the presence of corticosteroids. In contrast, addition of exogenous IL-2 did not correct the defect in lymphokine secretion. We believe that inhibition of the production of LMIF by steroid may lead to defective granulocytic function and thus, predispose to infection.

The effect of hydrocortisone on the production of gamma-interferon and other lymphokines by human peripheral blood mononuclear cells.

Concentrations of hydrocortisone as low as 0.08 microgram/ml significantly reduced the yields of gamma-interferon (IFN-gamma) when phytohemagglutinin (PHA) or concanavalin A (ConA) were used as inducers; however, when staphylococcal enterotoxin A was utilized, higher concentrations (5.0 micrograms/ml) were required to achieve the same effect. Yields of interleukin-2 (IL-2) and lymphotoxin were also found to be sensitive to the effects of the steroids, but expressions of TAC antigen was not generally affected by these agents. In contrast to the effects of steroids on cell proliferation, lymphokine production remained suppressed after steroid withdrawal. Hydrocortisone appeared to influence the concentrations of cyclic nucleotides following lectin stimulation, but attempts to correct these alterations or to add exogenous IL-2 failed to restore lymphokine production to normal levels. Addition of the calcium ionophore A23187 partially restored IFN-gamma production. We conclude that the effects of corticosteroids on the yields of lymphokines, including IFN-gamma, are profound. The depression of lymphokine production appears to be associated with a number of alterations in the cell, including depression of protein synthesis, alterations in cyclic nucleotides, and diminution of the production of cofactors necessary for IFN-gamma production. Enhancement of the flux of calcium into the cell may restore some of the ability to produce IFN-gamma.

Mondor's disease associated with metastatic axillary nodes.

Mondor's disease, superficial thrombophlebitis of the breast, is customarily associated with benign conditions of the breast. This article reports a patient in whom an early manifestation of recurrent axillary metastasis from carcinoma of the breast was a symptom of ipsilateral superficial thrombophlebitis of the breast, an unusual association.

Effect of antineoplastic agents on gamma-interferon production in human peripheral blood mononuclear cells.

Since gamma-interferon (IFN-gamma) is a potent immunomodulator and patients receiving certain antineoplastic agents are at risk of unusual infections, we have determined the effect of certain antineoplastic agents on IFN-gamma production. Induction of peripheral blood mononuclear cells from normal donors in the presence and absence of various antineoplastic agents was achieved using phytohemagglutinin (8 micrograms/ml). Supernatants were then separated by centrifugation, dialyzed, and assayed for interferon. Cell viability was always greater than 85% with or without the presence of drugs. Hydrocortisone was found to eliminate IFN-gamma production if added within 24 hr after the phytohemagglutinin. The suppression of IFN-gamma production occurred with hydrocortisone concentrations as low as 0.65 microgram/ml, was associated with a diminished proliferative response to the lectin, and occurred with other interferon inducers including staphylococcal enterotoxin A. Adriamycin (0.4 microgram/ml) and vincristine (0.08 microgram/ml) also diminished IFN-gamma production, but only if the peripheral blood mononuclear cells were pretreated with the drugs. Methotrexate, 5-fluorouracil, and 6-mercaptopurine failed to influence the yield of IFN-gamma. These results are significantly different from experiments previously reported using alpha- and beta-interferons and suggest an important mechanism by which these drugs can produce immunosuppression.

Bacterial meningitis in the elderly.

To assess the implications of meningitis in a more mature population, we reviewed the records of patients with meningitis: 71 aged 50 years and older and 138 patients aged 15 to 49 years. Among the older population, 54 (76%) had bacterial, nine (13%) had granulomatous, and eight (11%) had aseptic meningitis. Among the cases of bacterial meningitis in the older age group, Streptococcus pneumoniae accounted for 24% (13/54) and enteric bacilli accounted for 17% (9/54). Serious complications occurred in 38 elderly patients (70%) with bacterial meningitis, and mortality occurred in 24 (44%). In the younger age group with bacterial meningitis, the complication rate and mortality were 41% (13/32) and 13% (4/32), respectively. Meningitis in the elderly is likely to be bacterial and to cause greater morbidity and mortality.