Differentiation of Clostridium difficile, Clostridium bifermentans, Clostridium sordellii, and Clostridium perfringens from diarrheal stool by API ZYM and API LRA oxidase test.

A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed. It consists of a combination of API ZYM and API LRA Oxidase tests. The enzymatic activities were performed with strains sub-cultured onto carbohydrate-free medium (Columbia blood agar). Fifty-five strains of Clostridium difficile, C. bifermentans, C. sordellii, and C. perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed. The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.

Synergistic effect of thymosin alpha 1 and alpha beta-interferon on NK activity in tumor-bearing mice.

We have investigated the possibility of thymosin alpha 1 (TH) cooperating with alpha beta-interferon (IFN) in boosting natural killer (NK) activity in tumor-bearing, immunosuppressed mice in vivo. Treatment with a single injection of 30,000 IU of IFN 24 h before testing enhanced NK activity in tumor-bearing mice if the IFN was administered 9 days after tumor inoculation, when the animals have normal NK responsiveness. On the other hand, the same treatment led to lower or no improvement of NK responses if the treatment was given 13 or 17 days after tumor inoculation, at a time when tumor growth causes immunosuppression. However, combination treatment with TH (200 micrograms/kg) for 4 days, followed by IFN was found to restore normal NK cell activity. Selective depletion of antigen-positive cells showed that killer cells stimulated by combination treatment with TH and IFN seem to bear phenotypic characteristics of NK cells. These studies provide the first documentation of a novel combination approach to reconstitution of immunosuppressed tumor-bearing mice using TH and IFN. We hypothesize that TH restores NK boosting activity by IFN by effecting the differentiation/induction of precursor populations of IFN-responsive cells.

Effect of in vivo administration of prostaglandins and interferon on natural killer activity and on B-16 melanoma growth in mice.

We investigated the effect on in vivo administration of 16,16-dimethyl-prostaglandin E2 (di-M-PGE2) alone and in combined treatment with alpha-beta interferon (IFN) on NK activity in normal, cyclophosphamide (CY)-treated, tumor bearing or irradiated and bone marrow reconstituted mice and on B-16 melanoma growth. Normal mice inoculated with IFN (30,000 U/mouse, 24 hr before testing) showed a significant increase in NK activity, while those treated with di-M-PGE2 (10 micrograms/mouse daily X 4 days) showed a suppressed NK response. No difference was observed between mice treated with di-M-PGE2 alone and those treated with di-M-PGE2 associated with IFN. In immunosuppressed animals single treatments were slightly (di-M-PGE2) or not (IFN) effective, while the combined administration of di-M-PGE2 and IFN caused a marked increase in NK activity. Moreover, di-M-PGE2 was able to accelerate the recovery rate of NK activity in bone marrow-reconstituted murine chimeras, suggesting that the synergistic effect of prostaglandins and IFN could derive from the action of di-M-PGE2 on progenitor pre-NK cells and of IFN on effector cells. Finally, we observed a good correlation between the enhancement of the NK activity and the tumor growth inhibition.

Effect of thymulin on intracellular cyclic nucleotides and prostaglandins E2 in peanut agglutinin-fractionated thymocytes.

Prostaglandin E2 (PGE2) and other selected agents which elevate intracellular cyclic AMP (cAMP) levels have been demonstrated to induce the appearance of surface markers in immature T lymphocytes. Thymic hormones, which are the natural inducers of these markers, have long been hypothesized to act through the increase of cAMP levels. We have approached this area of investigation by studying the effects of thymulin (a serum thymus-derived factor, coupled with Zinc) on intracellular cAMP and cyclic GMP (cGMP) levels (expressed as cAMP/cGMP ratio) and on the release of PGE2 in different thymocyte subpopulations. Thymocytes were fractionated by the peanut agglutinin (PNA) technique into cortical immature PNA+ and medullary mature PNA- thymocytes. The data presented in this report show that thymulin is able to increase the cAMP/cGMP ratio in PNA+ and in unfractionated thymocytes, depending on its concentration, but not in PNA- thymic cells. Conversely, it is able to increase the release of PGE2 by PNA- thymocytes but not by PNA+ and unfractionated thymic lymphocytes. These results are consistent with the hypothesis that thymulin could act through different mechanisms depending on the differentiation stage of its target cells. In fact, it could be suggested that immature T cells could be activated by thymulin thereby increasing the cAMP/cGMP ratio, whereas more mature T cells would be further differentiated by thymulin through enhanced release of PGE2.

Receptors for thymosin alpha 1 on mouse thymocytes.

Thymosin alpha 1 is able to act in vitro to stimulate T-cell precursors and to induce surface markers. The initial mechanism by which alpha 1 activates T cells could be the binding of alpha 1 to cell membranes. Using a specific anti-alpha 1 antibody and an indirect immunofluorescence procedure it was found that thymosin alpha 1 binds to the surface of a large portion of murine lymphocytes. Furthermore, thymocytes have been fractionated into immature and mature subpopulations by using the peanut agglutinin (PNA) technique. It was found that PNA+, immature cells showed specific receptors for alpha 1 on the cell membrane.

Modulation of natural killer activity by thymosin alpha 1 and interferon.

A single injection of alpha beta-interferon (alpha beta-IFN) (30000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals. We investigated the possibility of thymosin alpha 1 cooperating with alpha beta-IFN in boosting NK activity in CY-suppressed animals. The results show that treatment with thymosin alpha 1 (200 micrograms/kg) for 4 days, followed by a single injection of alpha beta-IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin alpha 1 was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras. Taken together the data support the concept that the synergic effect between thymosin alpha 1 and alpha beta-IFN could be the result of effects on differentiation of the NK lineage at different levels.

Modulation of endogenous prostaglandins by thymosin-alpha 1 in lymphocytes.

The effects of thymosin-alpha 1 on the stimulation of specific release of prostaglandin E2 (PGE2) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-alpha 1 in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE2 between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-alpha 1 at certain concentrations was able to stimulate PGE2 release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE2 after incubation with alpha 1 in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentrations of alpha 1 was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of alpha 1 after administration of this synthetic molecule show a clear dissociation. A maximum peak of alpha 1 activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by alpha 1 of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of alpha 1 requires prostaglandin biosynthesis.

Modulation of natural killer (nk) cell activity during FLV-P virus infection of mice.

We analyzed the effects of a polycythemic substrain of Friend leukemia virus, i.e. the FLV-P virus, on splenic NK activity of DBA/2 susceptible mice. One day after virus injection a significant increase of NK activity was found, which persisted until day 10. On the other hand, 14-21 days after virus injection a marked and significant depression of activity was measured. This depression was associated with the appearance of suppressor cells able to inhibit the lytic activity of untreated splenocytes when mixed in vitro in the 4 h 51Cr-release assay. The suppressor cell population was insensitive to treatment with anti-Thy 1.2 plus complement, was adherent to Sephadex G-10 and nylon, but did not adhere to plastic, suggesting it is neither a T-cell nor a typical macrophage. The possible relevance of NK activity modulation in relation to the induction of leukemia is discussed.

Impairment of in vitro generation of cytotoxic or T suppressor lymphocytes by Friend leukemia virus infection in mice.

Spleen cells collected from DBA/2 (H-2d) mice inoculated with the polycythemic variant of Friend-Leukemia Virus Complex (FLV-P) were tested for T-dependent immune functions, such as the in vitro generation of cytotoxic T lymphocytes (CTL) and of non-specific T suppressor lymphocytes (STL). CTL were generated against H-2b splenocytes, and STL were obtained following a 5-day lymphocyte culture without stimulator cells. A progressive and severe impairment of the generation of both CLT and STL was found from 2 weeks onward after infection, being almost totally abolished 3-4 weeks after virus challenge. Suppressor cells (SC) capable of inhibiting CTL generation was detected in FLV-P bearing mice. Suppressor activity was unaffected by anti-Thy 1.2 serum and complement but was removed following iron-magnet depletion or passage through nylon-wool column. Moreover complete recovery of the competence of CTL generation was attained when FLV-P infected splenocytes were passed through nylon-wool column. It is concluded that FLV-P infection depresses T-dependent cytotoxic and suppressor responses in mice, by the appearance of non-T adherent phagocytic cells, capable of impairing CTL generation in vitro.

[Effects of PGA 1 on immune response in mice with melanoma B 16]. / Effetti delle PGA 1 sulla risposta immunitaria in topi porttori di melanoma B 16.

This study evaluated the effects of PGA1 on B 16 melanoma bearing mice. PGA1 was observed to inhibit the rate of melanoma growth, both as delay in the rate of appearance and decrease in tumor volume. Furthermore PGA1 stimulated humoral but not cellular immune response in melanoma bearing mice, while it was ineffective in normal mice.

[Preliminary study on the effects induced by an anemic strain of Friend leukemia virus in DBA/2 mice]. / Studio preliminare sugli effetti indotti dal ceppo anemico del virus della leucemia di Friend in topi DBA/2.

Serum thymic factor (STF) and azathioprine (AZ) inhibition test were assayed in sera and spleen cells from DBA/2 and BALB/c mice, inoculated with the anemic strain of Friend leukemia virus. The observed decrease of STF levels appears to be related to the LLV (Lymphatic Leukemia Virus) component of FLV-A.