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.

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.

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.

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.

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.

Nosocomial infection and antibiotic utilization in geriatric patients: a pilot prospective surveillance program in skilled nursing facilities.

Prospective surveillance of nosocomial infection was conducted at seven skilled proprietary nursing facilities in Orange County, Calif., USA. The average incidence of facility-acquired infection was 5.2 infections/1,000 patient days. The most common source of infection was urinary tract (47%), followed by respiratory tract (26%) and skin (14%). The four most common pathogens isolated were Proteus spp. (20%), Escherichia coli (17%), Staphylococcus aureus (13%) and Pseudomonas spp. (11%). Trimethoprim-sulfamethoxazole (20%) was the most frequently used antibiotic among all prescriptions, followed by ampicillin (16%) and ciprofloxacin (14%). Among all residents surveyed, 33% received at least one course of antibiotics during the study. Of special significance was the fact that 4 (22%) of the 18 strains of Pseudomonas were gentamicin resistant as were 12 of 80 (15%) of the strains of Enterobacteriaceae. Furthermore, 9 of 29 (31%) strains of Pseudomonas tested were found resistant to norfloxacin as were 15 of 129 (12%) strains of enterobacteriaceae. Susceptibility patterns of the isolated pathogens were similar to those of the acute care hospital. This study indicates that infection continues to be a major problem in the skilled nursing facility and that antibiotic-resistant pathogens will be a challenge for the future.

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.

Interleukin-1 and a 7-kDa T-cell inhibitory monokine reflect disease activity in infection with HIV-1.

Previous studies demonstrated that cultured peripheral blood mononuclear cells (PBMC) from patients with AIDS produce high levels of interleukin 1 (IL-1) and a 7-kDa T-cell inhibitory monokine (TCIM). To determine if the increase in the production of these cytokines corresponded with disease activity, we studied the production of IL-1 and TCIM by PBMC from patients with different stages of human immunodeficiency virus (HIV) infection. Eight patients with asymptomatic seropositive infection, three patients with AIDS-related complex (ARC), three patients with persistent generalized lymphadenopathy (PGL), and six patients meeting the full criteria for diagnosis of AIDS were studied. Patients with AIDS produced increased amounts of TCIM (4.1 times control values, p less than 0.003) and IL-1 (2.0 times control values, p less than 0.05). In contrast, asymptomatic seropositive patients produced less TCIM (0.36 times control values, p less than 0.004) and IL-1 (0.61 times control values, p less than 0.05). Different trends in the levels of these factors produced by patients with ARC and PGL were noted, although results were not statistically significant in general. Patients with ARC tended to produce less IL-1 (0.42 times control values, p less than 0.05), whereas patients with PGL tended to produce increased amounts of IL-1 (1.7 times control values, NS). ARC patients produced a wide range of TCIM values (0.05-2.8 times control values, NS), and patients with PGL tended to produce increased TCIM values, (4.0 times control values, p less than 0.02). No correlations between the levels of IL-1 or TCIM and T-cell subpopulation numbers (CD4 or CD8) or CD4/CD8 ratios were found.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Calcium and the production of interferon by human peripheral blood mononuclear cells.

We have studied the ability of human peripheral blood mononuclear cells (PBMC) to produce interferon-alpha (IFN-alpha) and IFN-gamma in the presence of pharmacologic agents known to influence calcium transport or calcium-dependent processes. We have found that the production of human (Hu) IFN-gamma is affected significantly by alterations in calcium flux; however, this influence is dependent upon the nature of the compound used to induce IFN. Inhibitors of protein kinase C decreased yields of IFN-gamma but inhibition of calmodulin did not. The presence of vitamin D3 reduced IFN-gamma titers when PHA and IL-2 were used to induce IFN, but not when ionomycin was used as the inducer. The production of IFN-gamma by PBMC was reduced by diminished concentrations in extracellular calcium but not extracellular magnesium. In contrast, neither the presence of any of the pharmacological agents tested above nor the reduction of the calcium concentration influenced the production of HuIFN-alpha by PBMC.

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.

Optimal conditions for the induction of human mononuclear leukocytes by complexed polyinosinic-polycytidylic acid.

We have defined the optimal circumstances for the induction of human interferon alpha (Hu IFN alpha) in peripheral blood mononuclear cells (PBMC) using complexed polyinosinic-polycytidylic acids (poly rIrC). We have found that a poly rIrC concentration of only 0.1 microgram m/ml or 100 fold less than commonly used in fibroblasts produced maximal yields. Similarly, we have found that the optimal concentration of DEAE-D was 250 micrograms m/ml. Only a minimum exposure period of 30 minutes to the poly rIrC-DEAE-D mixture was found necessary, but the temperature of this incubation was not found to be a critical variant. Finally, we found that addition of exogenous calcium to the inducing solution failed to influence the ultimate titers of the IFN produced.

Effect of acetyl salicylic acid on production and action of leukocyte-derived interferons.

Although acetylsalicylic acid does not itself induce interferon, acetylsalicylic acid was found to significantly enhance the production of both human alpha interferon and human gamma interferon when added with the appropriate inducers to cultures of human peripheral blood mononuclear cells. The mechanisms associated with this effect were investigated.

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.