Phagocytic cell death leads to enhanced release of pro-inflammatory S100A12 in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF) is a prototypical autoinflammatory syndrome associated with phagocytic cell activation. Pyrin mutations are the genetic basis of this disease, and its expression has been shown in monocytes, granulocytes, dendritic cells, and synovial fibroblasts. Pyrin functions as a cytosolic pattern recognition receptor and forms a distinct pyrin inflammasome. The phagocyte-specific protein S100A12 is predominantly expressed in granulocytes and belongs to the group of damage associated molecular patterns (DAMP). S100A12 can be detected at massively elevated levels in the serum of FMF patients, even in clinically inactive disease. Whether this is crucial for FMF pathogenesis is as yet unknown, and we therefore investigated the mechanisms of S100A12 release from granulocytes of FMF patients presenting clinically inactive. RESULTS: We demonstrate that FMF neutrophils from patients in clinical inactive disease possess an intrinsic activity leading to cell death even in exogenously unstimulated neutrophils. Cell death resembles NETosis and is dependent on ROS and pore forming protein gasdermin D (GSDMD), as inhibitors for both are capable of completely block cell death and S100A12 release. When pyrin-activator TcdA (Clostridium difficile toxin A) is used to stimulate, neutrophilic cell death and S100A12 release are significantly enhanced in neutrophils from FMF patients compared to neutrophils from HC. CONCLUSIONS: We are able to demonstrate that activation threshold of neutrophils from inactive FMF patients is decreased, most likely by pre-activated pyrin. FMF neutrophils present with intrinsically higher ROS production, when cultured ex vivo. This higher baseline ROS activity leads to increased GSDMD cleavage and subsequent release of, e.g., S100A12, and to increased cell death with features of NETosis and pyroptosis. We show for the first time that cell death pathways in neutrophils of inactive FMF patients are easily triggered and lead to ROS- and GSDMD-dependent activation mechanisms and possibly pathology. This could be therapeutically addressed by blocking ROS or GSDMD cleavage to decrease inflammatory outbreaks when becoming highly active.

Evidence that caspase-13 is not a human but a bovine gene.

Caspase-13 was reported to be a member of the human caspase family of proteases (Humke, E., et al., J. Biol. Chem. 273, 15702-15707, 1998). By contrast, a recent study (Lin, X., et al., J. Biol. Chem. 275, 39920-39926, 2000) could not confirm caspase-13 expression in human tissues. When we searched the GenBank database we found several expressed sequence tags (ESTs) from bos taurus completely matching the published caspase-13 sequence. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that bovine but not human peripheral blood mononuclear cells express caspase-13. From these cells we cloned two bovine caspase-13 splice variants and found that the sequence of the larger variant was identical to the mRNA published by Humke et al. Our findings strongly suggest that the previously published caspase-13 sequence is not of human origin but represents a bovine gene.

A technical solution for secondary arthritis due to chronic proximal tibiofibular joint instability.

Chronic instability of the proximal tibiofibular joint is an uncommon diagnosis and not frequently reported in the literature. The management options of this joint instability, complicated with secondary arthritis, have rarely been discussed and consist mainly of fibular head resection or arthrodesis of this joint. We describe a new technical procedure for addressing both the instability and the joint secondary arthritis. Stability of the joint is achieved by ligament reconstruction using a biceps femoris split passed through the tibial metaphysis and fixated back to the fibular head using bone anchors. The arthritic changes are addressed by interposition of a vascularized fascia lata strip. The described procedure offers a firm stabilization with no need for postoperative restrictions and an alternative to the inadvisable joint arthrodesis or resection.

Detection of cytomegalovirus DNA in CD34+ cells from blood and bone marrow.

Infection of hematopoietic progenitor cells with the human cytomegalovirus (HCMV) has been proposed as an explanation for the cytopenias associated with HCMV-related disease. To test this hypothesis, CD34+ cells, which include the hematopoietic progenitors, as well as mature leukocyte populations were purified on a fluorescence-activated cell sorter and analyzed for HCMV DNA by polymerase chain reaction (PCR). A total of 33 samples from 31 immunosuppressed as well as immunocompetent HCMV-seropositive individuals were studied. CD34+ cells were PCR-positive in four of seven bone marrow aspirates from allogeneic bone marrow transplant recipients, in three of eight aspirates from patients with acquired immunodeficiency syndrome, and in the first of two bone marrow samples from an immunocompetent patient with primary HCMV disease. CD34+ cells purified from peripheral blood for autologous and allogeneic transplantation were also analyzed, and 4 of 13 samples were HCMV DNA-positive. Interestingly, two of the four HCMV-positive samples were from healthy allogeneic donors. Among the mature leukocyte populations, the monocytes were most frequently found to be HCMV DNA-positive. No HCMV DNA was detected in the total bone marrow leukocytes of 13 healthy seropositive bone marrow donors or in the CD34+ cell fraction of three further seropositive donors. In conclusion, the data provide strong evidence that CD34+ hematopoietic progenitor cells can be infected with HCMV in immunosuppressed patients, while this cell population was not identified as a major viral reservoir in healthy HCMV-seropositive individuals.

Determination of TNF alpha, interferon alpha, interleukin 2 and reactivity in the leucocyte migration inhibition test in breast cancer patients.

To date, the results concerning the prognostic importance of parameters of cell-mediated immunity in breast cancer patients are very contradictory; moreover, in most of them the results are hardly comparable due to methodological differences and heterogeneous groups of patients. In 123 patients with nonmetastatic breast carcinoma TNF alpha, INF alpha, IL 2 and reactivity in the leucocyte migration inhibition test (LMI-Test) against autologous tumor tissue were determined and the results correlated with the clinical course of the disease up to a maximum of 108 months. In breast cancer patients TNF alpha-serum levels were significantly (p less than 0.05) elevated compared to healthy controls. We also found that patients with progressive disease had higher levels than patients without recurrences. There were no differences concerning the IL-2 and IFN alpha serum levels between cancer patients and controls, nor did we find a correlation with the clinical course of the disease. In 38% of all breast cancer patients examined, a MIF production against tumor tissue could be demonstrated in the LMI-test. There was no difference concerning the LMI-reactivity between the groups of lymph-node negative and positive patients, but the observation that those patients with an unfavourable clinical course respond more frequently with an enhanced macrophage migration and rarely with migration inhibition was considered of notable prognostic significance. According to these results, it is possible that determination of TNF alpha and delayed type hypersensitivity reaction against tumor tissue in the LMI-test is of clinical value for the determination of risk groups.

[Comparison of tumor-associated cellular and nonspecific humoral immune parameters in immunologic monitoring of breast cancer]. / Vergleich tumorassoziierter zellulärer und unspezifischer humoraler Immunparameter beim immunologischen Monitoring des Mammakarzinoms.

In 119 patients with breast cancer states pT1-4 N0-3 M0 the following parameters of tumor-associated cellular and unspecific humoral immunity were determined: (1) leukocyte-migration-inhibition test against preparations of the autologous and homologous tumor tissue; (2) determination of the immunosuppressive activity of the serum on the PHA-, Con A- and PWM-induced lymphocyte transformation; and (3) determination of the alpha-2 pregnancy-associated globulin (alpha-2-PAG) serum level. In both groups of lymph node negative and positive breast cancer patients, half of the patients showed a significant reaction in the LMI test in the form of a reduced or enhanced migration of the macrophages (enhancement). In lymph node negative breast cancer patients the immunosuppressive activity of the serum on the PHA- and CON A-induced lymphocyte reactivity was decreased, whereby we found a positive correlation between the immunosuppressive activity of the serum and the LMI reactivity against autologous tumor tissue. We found no differences in the alpha-2-PAG serum level between lymph node negative and positive breast cancer patients, but there were greatly increased alpha-2-PAG levels in LMI-reactive patients compared to those showing an enhancement in the LMI test. A tumor-associated cellular immunity could be shown in the LMI test in breast cancer patients, which can be correlated with other humoral immune parameters. Especially the demonstration of an immunological enhancement in the LMI test could possibly be used for the definition of high-risk groups.

Differences in lymphocyte mitogenic stimulation pattern depending on anaesthesia and operative trauma: I. Halothane-nitrous oxide anaesthesia.

Cell-mediated immunity (investigated by in vitro mitogen/antigen induced lymphocyte proliferation) is known to be depressed in the post-operative period. In the present investigation, performed with halothane/nitrous oxide inhalational anaesthesia in healthy patients without trauma (eye surgery) and with operative tissue trauma (gynaecological operations), only the combination of major surgery with halothane/nitrous oxide anaesthesia was associated with a depression of lymphocyte reactivity to phytohaemagglutin (PHA-P), Concanavalin A (Con A) and pokeweed mitogen (PWM). This lasted for 3-10 days post-operatively.

Differences in lymphocyte mitogenic stimulation pattern depending on anaesthesia and operative trauma: II. Combined neuroleptanaesthesia.

In this study, neuroleptanaesthesia reduced the reactivity of lymphocytes to phytohaemagglutin (PHA-P) and pokeweed mitogen (PWM) in patients in a manner similar to that seen with halothane/nitrous oxide and enflurane/nitrous oxide inhalational anaesthesia. These findings began 4 h post-operatively, lasted throughout the first post-operative day and were similar in both operative trauma groups (eye surgery/gynaecological surgery). However, unlike the situation with the inhalational agent, Concanavalin A (Con A)-induced proliferation did not alter. It was also noted that, on the third post-operative day in both trauma groups, PHA-P- and PWM-induced proliferation ratios were significantly higher than were the pre-operative values. These findings could be the result of a specific effect of neuroleptanaesthesia.

Occurrence and characterization of different types of cytotoxic antibodies in pregnant women in relation to parity and gestational age.

Lymphocytotoxic antibodies in sera from 55 pregnant women (of whom 23 were in their first, 21 were in their second, and 11 were in their third pregnancy), as detected by four different microcytotoxicity tests, were predominantly cold reactive and of the IgM class. During pregnancy, there is an increase in lymphocytotoxic antibody formation which is most pronounced after delivery. It is impressive that during first and second pregnancies antibody frequency is similar in the first and second trimesters, whereas in a third pregnancy there is a decrease intermittently. The behavior of antibody frequency in the second trimester may suggest a possible absorption by fetal and/or placental structures. The antibodies seem to be part of a natural immunization process and may play a protective role in pregnancy.

Serum interferon levels in patients having radiotherapy for cervical carcinoma.

The well-known stimulation of interferon (IFN) both by non-specific and immune-specific stimulants, the effect of IFN on tumor therapy and against radiation damage gave us reason to examine cervical cancer patients undergoing radiation therapy for the presence of IFN. IFN was found in several serum samples of only two out of 47 patients.

[The technic of "open pelviscopy". A new method for increased safety in laparoscopy]. / Technik der "Offenen Pelviskopie" Eine neue Methode zur Erhöhung der Sicherheit bei der Bauchspiegelung.

The method of "open pelviscopy" is described and compared with the common performance of gynecological pelviscopy. The "open pelviscopy" combines the advantages of laparotomy--preparation of anatomical layers under view of the operator--with those of pelviscopy--minimal traumatization and optimal view at the intraabdominal organs. This method avoids blind puncture of the abdominal cavity, which otherwise is necessary to perform the pneumoperitoneum and to use the optic instruments. Apart from the well established indications for diagnostic and therapeutic gynecological pelviscopy we prefer this method especially in obese patients and in those who formerly had several laparotomies.