Retrospective study of 14 cases of canine arthritis secondary to Leishmania infection.

OBJECTIVE: To describe the clinical appearance, laboratory findings and response to treatment of dogs with inflammatory joint disease associated with Leishmania infection. METHODS: Retrospective analysis of case records of dogs with serologically confirmed leishmaniasis and concurrent inflammatory joint disease presented between 2005 and 2011. RESULTS: In total, 14 cases met the inclusion criteria. Of these, five (36%) dogs were presented with monoarthritis, five (36%) with oligoarthritis and four (28%) with polyarthritis. The most frequently affected joint was the carpus. Both erosive and non-erosive disease was identified on radiographic examination. All dogs had an inflammatory synovial fluid with a high white cell count and a preponderance of neutrophils, and in eight (57%) cases Leishmania amastigotes were found in the synovial fluid smears. Dogs were treated with 50 mg/kg N-methylglucamine antimoniate twice a day for 1 month and 10 mg/kg allopurinol twice a day for 6 to 9 months combined with prednisolone in five cases. At the 6-month follow-up, eight (57%) dogs showed improvement in general and orthopaedic signs and four (28%) dogs were stable. CLINICAL SIGNIFICANCE: Leishmaniasis should be considered a differential diagnosis in dogs with inflammatory arthritis in endemic areas.

In vitro evaluation of the PEtU-PDMS material immunocompatibility: the influence of surface topography and PDMS content.

The aim of the present work is to evaluate the in vitro immunocompatibility of an elastomeric material with feasible applications in the cardiovascular field. In particular, since it is well known that surface chemistry and topography play a key role in the foreign body response, their influence on human monocytes was evaluated. The material, constituted by a poly(ether)urethane (PEtU) and a polydimethylsiloxane (PDMS), was synthesized to manufacture films and small-diameter vascular grafts with three different surface topographical features, smooth, rough and porous, and siloxane rates, 10, 30 and 40. Human THP-1 monocytes have been cultured for 72 h on the films and human blood has been circulating for 2 h into the grafts to assess leukocyte adhesion and cytokine releases. Materials extracts were utilized to evaluate monocyte apoptosis. Smooth films showed lower cell adhesion degrees than rough and porous ones. All the PEtU-PDMS (poly(ether)urethane-polydimethylsiloxane) films and vascular grafts induced a narrow inflammatory response, as demonstrated by slight cytokine secretion levels, in particular samples with the highest PDMS contents (30 and 40%) induced the lowest IL-1b secretion. Moreover, an absence of monocyte apoptosis advises that the negligible release values have not to be ascribed to material toxicity. In the end, surface topography showed to affect only monocyte adhesion while siloxane content the cytokine release. Therefore, the possibility to modify the above tested parameters during material synthesis and manufacture could allow to bound the inflammatory potency of the PEtU-PDMS devices and render them excellent candidates for cardiovascular reconstruction.

Reduction of blood coagulation and monocyte-platelet interaction following the use of a minimal extracorporeal circulation system (Synergy) in coronary artery bypass grafting (CABG).

Cardiovascular surgery with cardiopulmonary bypass (CPB) induces activation of blood coagulation and systemic inflammation involved in post-operative complications. Our study evaluated the impact of the minimal extracorporeal circulation (mini-CPB) system (Synergy, Sorin Group) on these functional aspects. Twenty patients were randomly assigned to standard CPB (n = 10) or to Synergy (n = 10). Platelet expression of PAC-1, and monocyte/granulocyte-platelet conjugates were evaluated by flow cytometry. A leukocyte-platelet adhesion index was calculated after cell number normalization. ELISAs were performed to measure IL-6 and TNF-alpha, thrombin-antithrombin III complexes (TAT), prothrombin fragments (F1+2), beta-thromboglobulin (beta-TG) and sP-selectin (sCD62P). Blood samples were drawn at the time of anesthesia (T1), at the end of CPB (T2), and at 4 (T3) and 24 hours (T4) after weaning from CPB. All patients were similar for clinical characteristics. When compared to standard CPB, the Synergy showed lower levels of the monocyte-platelet adhesion index at T2 (0.023 +/- 0.005 vs 0.063 +/- 0.013, P = 0.0092) and T4 (0.031 +/- 0.003 vs 0.055 +/- 0.005, P = 0.0017), TAT complexes at T2 (27.175 +/- 5.967 vs 86.592 +/- 5.415, P = 0.0005) and T3 (26.977 +/- 2.468 vs 45.146 +/- 4.365, P = 0.0041), F1+2 fragments at T2 (2.222 +/- 0.226 vs 4.249 +/- 0.292, P = 0.0009), and sP-selectin at T3 (115.17 +/- 19.623 vs 169.554 +/- 19.709, P = 0.0703) and T4 (108.542 +/- 6.429 vs 140.799 +/- 14.771, P = 0.0833). In summary, the Synergy exhibited a lower post-operative activation of blood coagulation, together with a reduced interaction between circulating monocytes and platelets.

An alternative and simple method to consistently prepare viable isolated human islets for clinical transplantation.

We describe a method to consistently prepare human islets for transplantation. By combining a simple collagenase digestion method and a density gradient purification system, we were able to obtain successful isolations (>/=200,000 islet equivalents, >/=50% purity) in 69% of processed glands. No reagent of animal source was used. Isolated islets were morphologically well maintained and functionally competent, with sterility confirmed in 97% of cases. Two patients were transplanted with islets prepared by this method; graft function was demonstrated for a few months. Improved simplicity and consistency, together with adequate quality of the preparations, are the main features of this isolation method.

Purpurin-18 in combination with light leads to apoptosis or necrosis in HL60 leukemia cells.

Photodynamic therapy (PDT), a cancer treatment using a photosensitizer and visible light, has been shown to induce apoptosis or necrosis. We report here that Purpurin-18 (Pu18) in combination with light induces rapid apoptotic cell death in the human leukemia cell line (HL60) at low doses and necrosis at higher concentrations. Cells treated with Pu18 and light under apoptotic conditions exhibited DNA laddering and an increase in both cellular content of subdiploid DNA and externalization of phosphatidylserine (PS), indicating DNA fragmentation and loss of membrane phospholipid asymmetry. In the absence of light activation, Pu18 at nanomolar concentrations had no detectable cytotoxic effect. Caspase-3 activity was increased even after 1 h from treatment with low doses of Pu18 and light. The PS exposure and nuclear features of apoptosis were prevented by treatment of cells before illumination with caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK). Conversely, the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) failed to suppress the apoptosis. No protective effect of the three caspase inhibitors was observed when the cells were exposed to necrotic concentrations of Pu18 and light. Our results show that caspase-3, but not caspase-1, is involved in the signaling of apoptotic events in PDT with Pu18-induced apoptosis of HL60 cells. Moreover, both the time course of PS exposure and the effect of caspase inhibitors on it indicate that it is regulated in the same manner as DNA fragmentation.

Inhibitory effects of human neutrophil functions by the 45-kD glycoprotein derived from the parasitic nematode Trichinella spiralis.

AIM: We evaluated the effect of the 45-kD protein of Trichinella spiralis (gp45), purified by affinity chromatography, on random migration and chemotaxis, the oxidative metabolism of human neutrophils and on the CD11b upregulation induced by formyl-methionyl-leucyl-phenylalanine (f-MLP). METHODS: Donor neutrophils incubated with different amounts of gp45 (0.5, 1, 1.5, 2 microg/ml) or buffer and the random migration and chemotaxis, evaluated by means of a special technique of image analysis, and the chemiluminescence response to f-MLP or phorbol myristate acetate (PMA) were analyzed. The effect on CD11b upregulation was assessed incubating cells with the protein, when activating them with f-MLP. RESULTS: The results showed that gp45 inhibited both random and stimulated migrations, and reduced the response to f-MLP and PMA. Furthermore, gp45 significantly reduced the upregulation of the CD11b induced by f-MLP. CONCLUSION: The results show that gp45 inhibits PMN in different functions, suggesting an anti-inflammatory action.

Immunological alterations in adult obsessive-compulsive disorder.

BACKGROUND: Some recent findings suggest the involvement of autoimmune mechanisms in childhood onset of obsessive-compulsive disorder (OCD), on the basis of a parallel drawn with Sydenham's chorea, a manifestation of rheumatic fever. A monoclonal antibody called D8/D17 characterizing a B-lymphocyte antigen, present in almost all patients with rheumatic fever, has been found also in children affected by OCD, Tourette syndrome, and chronic tics to a greater degree than in healthy control subjects. The few observations of disturbances of some immunologic parameters in adult OCD patients, prompted the authors to investigate and compare subsets of peripheral immunological cells for differences in adult patients with OCD and healthy control subjects. METHODS: Twenty patients suffering from OCD, with no comorbidity for other psychiatric disorders, were compared with a similar group of healthy control subjects. The immune subsets were measured by flow cytometry. RESULTS: The CD8+ lymphocytes were significantly increased and CD4+ lymphocytes significantly decreased in OCD patients, while the other cells did not differ between the two groups. No correlation was found between immunologic and clinical parameters. CONCLUSIONS: These data indicate that patients with adult OCD showed increased CD8+, i.e., suppressor T lymphocytes, and decreased CD4+, which identify helper T lymphocytes, as compared with a similar group of healthy control subjects. The findings appear peculiar to patients with OCD and are suggestive of an immunologic imbalance, which might be related to the stress deriving from the frustrating situation determined by the disorder itself.

Detection of eosinophils in whole blood samples by flow cytometry.

A flow cytometric method to detect and study human eosinophils in whole blood was established. Normal subjects and patients with various types of eosinophilia (hypereosinophilic syndromes, allergic diseases, dermatitis, Hodgkin's Disease, parasitosis) were studied. Whole blood samples were treated for 10 minutes at room temperature with a commercially available reagent (FACS Lysing Solution, Becton Dickinson) which acts both as a fixative and as a lysing agent. Eosinophils were identified as a granulocytic subpopulation with higher SSC and FSC properties. This cell population was characterized by evident autofluorescence and hypodiploid DNA features after propidium iodide staining. The purity of the eosinophil population sorted after electronic gating was close to 100%. A very significant correlation between eosinophil counting by our whole blood method and other two assays, namely routine automatic counting by the H*3 Bayer System and eosinophil detection by depolarized SSC, was obtained. The phagocytic properties of eosinophils were also studied by means of a commercially available diagnostic kit, thus demonstrating that our method is also suitable for the study of those granulocytic functions which can be evaluated by flow cytometry.

fMLP-induced CD11b/CD18 upregulation on neutrophils from patients with non-Hodgkin's lymphomas treated with recombinant human granulocyte colony-stimulating factor.

The effects of rhG-CSF administration on fMLP-induced neutrophil CD11b and CD18 upregulation were studied in nine patients suffering from intermediate and high grade non-Hodgkin's lymphomas. Blood samples were obtained before recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration and 24 hrs after rhGSF interruption. The growth factor was administered subcutaneously for five days in a dosage of 5 microg/Kg/day. Nine normal subjects were studied as controls. Five patients showed an impaired baseline CD11b and CD18 upregulation, which was corrected by rhG-CSF therapy. Four patients showed a normal baseline CD11b and CD18 upregulation, but this function was reduced by rhG-CSF therapy. All patients showed a normal baseline fMLP-induced luminol-enhanced chemiluminiscence and significantly increased chemiluminescence values after rhG-CSF administration. We conclude that, while in some patients rhg-CSF is able to improve neutrophil CD11b and CD18 upregulation in response to chemotactic agents, in other patients a decrease of this function can occur, maybe due to a relative immaturity of the circulating neutrophils induced by rhG-CSF.

Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes--a flow cytometric study.

In this study F-actin polymerization in neutrophils from 21 patients affected by myelodysplastic syndromes (MDS) was evaluated by means of a flow cytometric assay. Neutrophils were stimulated with formyl-methionyl-leucyl-phenylanaline (fMLP; 10(-8) M final concentration) for 15, 30, 60 and 120 sec, and F-actin content was determined using fluorescein-isothiocyanate phallacidin as a specific probe. Eight normal subjects were studied as controls. We found that F-actin polymerization was defective in ten patients, with very impaired values after 60 and 120 sec of stimulation with fMLP. The remaining 11 patients showed a prevalent neutrophil population with normal F-actin polymerization and neutrophil sub-populations with either defective or undetectable F-actin polymerization. In the first group, patients with very poor prognosis (refractory anemia with excess blasts, refractory anemia with excess blasts in leukemic transformation, trisomy 8, multiple karyotypic abnormalities) were present, although patients with aberrations of karyotype were present in the second group. It is possible that defects in neutrophil F-actin polymerization may be responsible for neutrophil dysfunction, which has frequently been observed in MDS.

Granulocyte colony-stimulating factor (G-CSF) administration increases PMN CD32 (FcRII) expression and FcR-related functions.

The phenotypical and functional properties of circulating neutrophils from ten patients suffering from intermediate- and high-grade non-Hodgkin lymphoma were investigated before and after rhG-CSF administration (5 micrograms/kg/day subcutaneously for 5 days). The following parameters were studied: flow cytometry evaluation of surface CD32, CD16, CD11b and CD18 by means of a whole blood method; whole blood phagocytosis by means of a flow cytometric assay; whole blood chemiluminescence using opsonized zymosan as a stimulus. A significant increase in the expression of surface CD32 was detected in all patients, while CD11b expression was found to be increased in only four of them. CD16 and CD18 expression did not change. A significant enhancement of phagocytosis and phagocytosis-associated chemiluminescence was also observed. These results show that rhG-CSF administration can increase both FcRII expression and FcR-related functions.

Effects of lysine-arginine association on immune functions in patients with recurrent infections.

Combined L-lysine-L-arginine therapy is capable of inducting recovery in age-related decline of thymic activity in mice and in elderly humans. The clinical usefulness of the association has also been shown in children with recurrent respiratory infections, while an increase in the number of CD3+ lymphocytes has been shown in patients with chronic lymphatic leukaemia. Recently, in vitro effects of the association on neutrophil function have been reported. In particular, the association was able to increase random migration, chemotaxis, phagocytosis-associated- and f-MLP-induced chemiluminescence. In this paper the authors evaluate the effects of L-lysine-L-arginine combination (lisargin) on several humoral and cell-mediated immunologic parameters in patients with recurrent infection. An increase of neutrophil random migration and chemotaxis (evaluated by a new technique, based on a computer assisted image processing system) was found. Furthermore an increase in the absolute number of lymphocytes involved in cytotoxic activity and IgG levels was observed.

Lysine-arginine association and human neutrophil function: enhancement of functions related to microbicidal activity.

The effects of L-lysine, L-arginine, and lysine-arginine association on human neutrophil function were studied. The combination of the two agents, used at pharmacological concentrations, was able to increase both the phagocytosis-associated and the f-MLP-induced chemiluminescence, in the presence of luminol as amplifier agent. Neither superoxide anion production nor phytohaemagglutinin-induced aggregation were affected by the lysine-arginine association. The two amino-acids did not show any effect when used as single agents. L-canavanine (competitive inhibitor of nitric oxide synthesis starting from L-arginine) did not reduce chemiluminescence. These results show that the association lysine-arginine may increase the microbicidal potential of human neutrophils, probably by increasing the production of hypochlorous acid. In addition, a role for nitric oxide in these effects can be excluded.

Mycobacterial infection in HIV+ patients detected with a new immunological method (TB test).

Antibody response to antigen A60 (a mycobacterial antigen) was evaluated in ELISA in 18 HIV+ subjects with clinical and cultural evidences of mycobacterial infections, in 10 HIV+ subjects without Mycobacterial infections and in 22 healthy donors. We found higher levels of specific IgG in the HIV+ patients with Mycobacterial infections (mean 179.2 +/- 83 U) compared to the values of the donors (mean 92.5 +/- 35.5 U: p < 0.01). This test may be useful in the diagnosis of tuberculosis, but it needs clinical validation.

In vitro effects of an immunoglobulin preparation for intravenous use (IVIG) on T-cells activation.

The "in vitro" effect of different concentrations of polyclonal human immunoglobulins for intravenous use (IVIG) on cultured T-lymphocyte response to phytohemagglutinin (PHA) was evaluated. Four dilutions of IVIG corresponding with the "in vitro" doses currently employed in therapy have been tested by the effect on PHA stimulated cultures. The dilution of IVIG corresponding with a low therapeutical dose gives a higher statistically significant stimulating effect. The results obtained in our work agree with previous studies performed on activated B-cells. We can suppose the immunoglobulins may control the T-lymphocyte activation either by a direct link with a specific Fc-receptor expressed on the surface of activated T-cell or by affecting the cytokines release from monocyte cells.

IgE modification of the specific antidermatophagoides during the first year of specific immunotherapy (SIT).

Six subjects (4 female, 2 male), aged from 16 to 25 years, presented with allergic rhinitis to Dermatophagoides mites and received SIT by the sub-cutaneous route with delayed-release alpha fraction Bayropharm at the standard doses. Diagnosis was based on clinical history, skin tests and measurement of specific IgE at 0, 3, 9, and 12 months, by the fluoro-enzymatic technique (FAST). For comparison, in a reference group (n = 20) the IgE varied between 0.32 and 0.11 IU/ml for D1 and 0.31 to 0.09 IU/ml for D2. The eight patients had specific IgE titres of D1 = 0.96, D2 = 0.99. For these authors, the FAST technique used for the measurement of specific IgE, although less sensitive than the RIA technique of RAST, gives a good evaluation of SIT.