The mediator of cellular immunity. IX. The relationship between cellular hypersensitivity and acquired cellular resistance in rats infected with Listeria monocytogenes.

Acquired resistance to the intracellular bacterial parasite, Listeria monocytogenes can be transferred to normal recipients by thoracic duct lymphocytes or peritoneal exudate cells obtained from rats infected with this organism; The appearance of protective cells in thoracic duct lymph coincides with the development in the donors of delayed-type hypersensitivity to Listeria antigens and accumulation in induced peritoneal exudates of cells which are responsive to these antigens in the migration inhibitory factor (MIF) assay. The cells in exudates that confer protection, and those that release MIF, arise at sites remote from their final destination. From their point of origin in the caudal lymph nodes of infected rats, cells with these properties are delivered to the thoracic duct and hence to the blood from where they are drawn into the peritoneal cavity in response to inflammation. The parallel observed in the appearance, increase and subsequent decline of protective lymphocytes and MIF-producing cells in exudates suggest that the two activities are mediated by a single line of T cells. However this may be, the development and deployment of the cells concerned encourages the belief that MIF has a meaningful role in the expression of cellular resistance to infection.

Immunological and ultrastructural characterization of true histiocytic lymphoma in the northern pike, Esox lucius L.

An epidemic of a malignant neoplasm occurs in northern pike, Esox lucius L., from the Aland Islands of Finland. The neoplasm is morphologically similar to other pike hemic tumors reported in other areas of the world. Pike normal tissues showed evolutionary conservation with the mammalian intermediate filament proteins cytokeratin, desmin, vimentin, neurofilament protein, and glial fibrillary acidic protein; tumor cells are positive for vimentin, suggesting that the neoplasm is of mesenchymal origin. Hemic tissue mononuclear cells undergo polyclonal stimulation by the known mammalian T- and B-lymphocyte mitogens phytohemagglutinin P, concanavalin A, tuberculin-purified protein derivative, and lipopolysaccharide W; pike tumor cells are nonreactive. Pike normal hemic tissue mononuclear cells are variously positive for surface and cytoplasmic immunoglobulins, using rabbit anti-pike immunoglobulin M and cross-reactive mouse anti-carp immunoglobulin M antibodies; tumor cells, however, are not positive. The tumor cells were also diffusely stained with sodium fluoride-sensitive nonspecific esterase. The foregoing suggest that the neoplasm is not of B-lymphocytic or plasmacytic derivation, while the T-lymphocytic as opposed to monocytic derivation cannot be excluded on the basis of marker studies. The ultrastructural studies, however, suggest a neoplasm of histiomonocytic derivation, while the absence of sinusoidal infiltration of tumor cells to head kidney, spleen, liver, or peripheral blood suggests that it is a piscine analogue of human true histiocytic lymphoma. Population dynamics studies indicate that the neoplasm affects primarily sexually mature males 5 to 6 yr of age, but does not at present appear to be a major factor affecting Aland pike populations.

Nosocomial bloodstream infections in Finnish hospitals during 1999-2000.

Prospective laboratory-based surveillance in 4 Finnish hospitals during 1999-2000 identified 1477 cases of nosocomial bloodstream infection (BSI), with an overall rate of 0.8 BSIs per 1000 patient-days. Of BSI cases, 33% were in patients with a hematological malignancy and 15% were in patients with a solid malignancy; 26% were in patients who had undergone surgery preceding infection. Twenty-six percent of BSIs were related to intensive care, and 61% occurred in patients with a central venous catheter. Sixty-five percent of the 1621 causative organisms were gram positive, 31% were gram negative, and 4% were fungi. The most common pathogens were coagulase-negative staphylococci (31%), Escherichia coli (11%), Staphylococcus aureus (11%), and enterococci (6%). Methicillin resistance was detected in 1% of S. aureus isolates and vancomycin resistance in 1% of enterococci. The 7-day case-fatality ratio was 9% and was highest for infections caused by Candida (21%) and enterococci (18%). The overall rate of nosocomial BSIs was similar to rates in England and the United States, but S. aureus, enterococci, and fungi were less common in our study, and the prevalence of antibiotic resistance was lower.

Cutaneous tumor of northern pike, Esox lucius L. II. Expression of surface and cytoplasmic immunoglobulins and alpha-naphthyl acetate esterase by mononuclear cells from lymphoid tissues and tumor.

Rabbit anti-pike IgM antibodies showed that up to 95% of mononuclear (MN) cells isolated on Ficoll-Isopaque gradients from peripheral blood, spleen, and head kidney of northern pikes carrying cutaneous tumors were surface (SIg) and cytoplasmic (CIg) immunoglobulin positive by indirect immunofluorescence. On the contrary, rabbit anti-winter flounder IgM antibodies gave totally negative results. Monoclonal anti-carp IgM antibodies stained approximately 40-56% of peripheral blood, splenic, and head kidney MN cells in assays for SIg and CIg. Monoclonal anti-rainbow trout IgM antibodies, however, did not cross-react with pike Ig. A maximum of 5% of MN cells from the tumor were SIg and CIg positive by anti-pike IgM as well as 6% by anti-carp IgM antibodies, and were negative by the other two antibodies even at low dilutions. A majority of cells in the tumor exhibited an intense diffuse staining pattern for alpha-naphthyl acetate esterase (ANAE) which was inhibited by sodium fluoride and was reminiscent of that noted in human monocytes. These findings show that the Aland pike neoplasm is not a B cell tumor or plasmacytoma, but more representative markers are needed to demonstrate whether it is of T lymphocytic or monocytic origin.

Cutaneous tumor of northern pike, Esox lucius L. I. In vitro mitogenic responses of mononuclear cells from lymphoid tissues and tumor.

To evaluate functional characteristics of lymphocytes from the northern pike, Esox lucius L., mononuclear cells were isolated by Ficoll-Isopaque gradients from lymphoid organs and cutaneous tumors of normal and tumor bearing pikes. The cells were tested in a lymphocyte proliferation assay in medium supplemented with fetal calf serum or autologous plasma using three concentrations of PHA, Con A, tuberculin PPD, and LPS. Lymphocytes of northern pike could be triggered to DNA synthesis in vitro. However, no clearcut anatomical partitioning of mitogen responses was found, since the mean optimal proliferation indices for each mitogen were similar in blood, head kidney, and spleen, while cells from head kidney, the equivalent of bone marrow, were stimulated as well with the murine T cell mitogens PHA and Con A as with the B cell mitogens PPD and LPS. Tumorous pikes seemed to have an apparently normal lymphocyte population since they responded by blastogenesis as well as normal pikes. Tumor cells exhibited a high basic metabolic rate and reactivity to mitogens was largely lacking.

Detection of experimental bacteremia and fungemia by examination of buffy coat prepared by a micromethod.

Rabbits received intravenous injections of bacteria or fungi, and a comparison was made of the abilities of broth cultures, plating after dilution either in saline solution or in distilled water containing Triton X-100, and buffy coat examinations to detect the organisms in heart blood. The most sensitive method was broth culture. By microscopy or subculture of buffy coat cells prepared by centrifugation of blood in microhematocrit tubes, organisms were rapidly and regularly detected when their viable counts increased to 300--1,000/ml as detected by plating. By micromodification, buffy coat examination is technically easy to perform, and the method is only slightly less sensitive than when a larger amount of blood is used. Thus, it would be ideal for rapid provisional diagnosis of sepsis in patients, e.g., neonates, when the use of only a small blood sample is preferred.

Dipslide cultures in the investigation of suprapubic urinary bladder aspirates of infants and children.

Suprapubic aspirates (SPAs) of the urinary bladder obtained from 793 infants and children with suspected urinary tract infection were grown on dipslides inoculated at the bedside as well as being cultured aerobically and anaerobically. In general, the findings on dipslides were in good agreement with those in the other culture media. The majority of the 403 dipslide-positive samples contained 10(5) or more bacteria/ml; only in 2% was bacterial density less than 10(3)/ml. There were five specimens with no growth on dipslides; however, an organism considered to be significant for the patient was isolated in the other cultures. Thus dipslides proved to be adequate for the culture of most SPAs, but samples from patients with urological problems should be studied by more complete methods.

Pseudomonas aeruginosa infection and vaccination in the rat.

A large molecular-weight fraction of Pseudomonas aeruginosa culture filtrate protected rats and mice against a lethal infection with a heterologous serotype, and to some extent against Escherichia coli and Listeria monocytogenes. The active components were obtained from cultures grown for several days in a simple synthetic medium. Infection and vaccination with P. aeruginosa serotype 16 induced agglutinating and precipitating antibodies to the components of this serotype only; in rats infected or vaccinated with serotype 1, low titres of agglutinating antibody against type 16 were found. Vaccine prepared from type 1 or 16 increased, within 3 days of infection, the resistance of rats to type-16 organisms; within the same period agglutinins or precipitins were not produced. It is possible that the protection was based on opsonic and antitoxic activities.

Susceptibility of fungi in mouthrinse specimens from patients with haematological malignancies.

Fungi isolated from mouthrinse specimens during episodes of acute pseudomembranous fungal stomatitis and deep-seated mycoses in patients with haematological malignancies were tested for susceptibility to seven antifungal agents. Topical treatment of stomatitis with clotrimazole or chlorhexidine did not induce any change in the susceptibility of oral Candida albicans. Treatment of deeper mycoses with 5-fluorocytosine, however, resulted in a significant increase in oral strains resistant to this agent. Of C. albicans strains isolated, 7% were resistant to 5-fluorocytosine greater than 32 micrograms/ml. One patient died of disseminated mycosis during treatment with this drug; the resistant C. albicans was isolated from the mouth, liver, spleen and kidneys. Strains of Torulopsis glabrata and C. krusei resistant to 5-fluorocytosine were also found in some patients. Organisms resistant to 5-fluorocytosine were generally sensitive to polyenes and imidazoles.

Antibodies against antigens of Candida albicans in patients with fungaemia and bacteraemia, studied by ELISA, precipitation, passive haemagglutination and immunofluorescence techniques.

Antibodies against commercially available antigens of Candida albicans were assayed in 54 sera from 24 patients with fungaemia and in 66 sera from 33 patients with bacteraemia. In patients with persistent fungaemia, antibody was found during the week after the fungus was first cultured from the blood, but peak titres did not usually occur until the end of the second week. A significant rise in titre in C. albicans infection was observed in 50% of paired sera tested by passive haemagglutination (PHA), indirect immunofluorescence (IF) and Ouchterlony immunodiffusion (ID). The same percentage was obtained by counterimmunoelectrophoresis (CIE) against candida metabolic antigens, whereas it was increased to 88% when somatic antigens were used. Enzyme-linked immunosorbent assay (ELISA) demonstrated a rise of titre in 25, 75 and 50% of sera in IgM, IgG and IgA assays, respectively. Sera from patients with transient fungaemia demonstrated persistent antibody titres. In paired sera from patients with bacteraemia, ID and CIE titres were low (greater than or equal to 4). There was an increase of candida antibodies in 0-9% of patients by ELISA, ID or CIE and in 18-21% by PHA or IF. Clinically significant fungaemia was most reliably differentiated serologically from bacteraemia by CIE S-antigen and ELISA IgG assays.

Immunological classification of a pike lymphoma.

Monoclonal and polyclonal antibodies against mammalian intermediate filament (IF) proteins were used to demonstrate IF expression in tissues and lymphoma tumors of northern pikes, Esox lucius L., from the Aland Islands of Finland by indirect immunofluorescence microscopy. Frozen sections of pike tissues demonstrated IFs in a manner confirming their evolutionary conservation and subclass specificity. Tumor cells showed morphological resemblance to head kidney cells and were positive for vimentin while negative for cytokeratin, desmin, neurofilament proteins, and glial fibrillary acidic protein. The results show that the neoplasm is a mesenchymal as opposed to an epithelial, muscle, neural, or glial tumor, and is probably hemic cell derived. A rabbit anti-pike IgM antiserum showed that up to 90% of mononuclear (MN) cells isolated on Ficoll-Isopaque gradients from peripheral blood, spleen and head kidney were surface- and cytoplasmic-immunoglobulin positive by indirect immunofluorescence, while a maximum of 5% of tumor cells were positive. A maximum of 5% of MN cells from hemic tissues exhibited rosettes when incubated with AET-treated sheep red blood cells; however, only 1% of cells in the tumor formed rosettes. Lymphocyte proliferation assays were performed on MN cells from hemic tissues and tumor using phytohemagglutinin P, concanavalin A, tuberculin purified protein derivative and lipopolysaccharide W in medium supplemented with fetal calf serum or autologous pike plasma. Proliferation indices in hemic tissues were similar in the groups. However, all proliferation indices in tissues were significantly higher than corresponding values in the tumor. These assays show that pike MN cells respond when stimulated by T and B cell mitogens, but that this reactivity is lacking in the tumor.

Delayed-type hypersensitivity measured as an increase of ear thickness in guinea pigs.

Determination of swelling at an intracutaneous test site in the pinna of the ear of guinea pigs immunized with protein antigens in complete Freund's adjuvant was found to be a more sensitive assay of delayed-type hypersensitivity than the measurement of flank skin erythema and induration. In fact, 100 times less specific antigen was needed to detect 24-hour reactivity in the pinna of the ear. Reactivity early after sensitization (cutaneous basophil hypersensitivity), however, was best revealed as an erythematous lesion of the flank skin 24 h after testing.

Radiometric ear index test as a measure of delayed-type-hypersensitivity in the rat.

Intradermal skin tests performed in the pinna of the rat ear appeared to be 100 times more sensitive than classical flank skin tests in measuring Arthus and delayed-type hypersensitivity (DTH) reactions. One of these tests was antigen-induced thickening of the pinna of the ear. It was found to be a sensitive measure of Arthus reactivity at 4 h after irritation with antigen in both actively immunized rats and recipients of precipitating immune serum. The other test, radiometric ear index determination, exploits the fact that monocytes and monocyte derived macrophages accumulate at DTH reaction sites. The test was performed by labelling the precursors of these cells with a pulse of [3H]-thymidine and by determining radioactivity in biopsy specimens taken from test sites in the pinna of the ear. At a certain antigen dose range this objective and highly sensitive method was shown to measure a purely cell mediated reaction which could be transferred to normal recipients with thoracic duct lymphocytes but not with immune serum. It also behaved as a typical DTH reacttion in response to desensitizing injections of the specific antigen. Testing with unnecessarily high antigen doses, however, should be avoided since the strong early inflammation induced by them may interfere with the determination of DTH while using this sensitive assay.

Serum levels of C-reactive protein in patients with pulmonary tuberculosis and malignant tumors of the chest.

Serum C-reactive protein (CRP) concentration was studied in patients with newly diagnosed post-primary pulmonary tuberculosis and in those with malignant intrathoracic tumors. In tuberculosis, there was a wide scatter in CRP values and the mean did not differ from that of the tumor patients. Tuberculous patients with cavitation in chest X-ray had significantly higher levels of CRP than those without as well as healthy controls. Normal CRP did not exclude tuberculosis and all the values were below 100 mg/l.

Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods.

An extract of Candida albicans was used as an antigen on microtitre plates in the enzyme-linked immunosorbent assay (ELISA) to measure IgM, IgG and IgA class antibodies in the sera of hospitalized patients. It was found that of these patient sera that reacted positively in Ouchterlony immunodiffusion (ID) when undiluted, 58% were also positive in the ELISA against the same antigen preparation. However, all the sera with an ID titre of 1:2 or higher were ELISA-positive, demonstrating especially IgG and IgA. Of the sera positive by counterimmunoelectrophoresis against somatic and metabolic antigens of C. albicans, 86% were positive by ELISA. Reactions in precipitin-negative sera, if they occurred, usually demonstrated IgM or IgA. The sera with high passive haemagglutination or indirect immunofluorescence titres against surface antigens of C. albicans were positive in the IgG and IgA assays, while approximately one third were positive in the IgM assay.