Winfried Padberg

Development of suppressor lymphocytes during acute rejection of rat cardiac allografts and preservation of suppression by anti-IL-2-receptor monoclonal antibody

Suppressor acivity was investigated in rats undergoing acute rejection of heterotopic cardiac allografts. Spleen cells were harvested at 7 days from LEW rats rejecting (LEWxBN) F1 heart grafts and fractionated into their T, T suppressor/cytotoxic, and t helper subpopulations. Transfer of alloimmune unseparated spleen

Synergy between subtherapeutic doses of cyclosporine and immunobiological manipulations in rat heart graft recipients.

Cyclosporine in combination with other chemical or biological immunosuppressive modalities has been useful in clinical and experimental organ transplantation. In these studies, the efficacy of adjunctive subtherapeutic doses of CsA given to immunologically enhanced heart graft recipients or to animals treated with an anti ‐IL‐2 receptor monoclonal antibody (ART18) are described. Individually, the treatment entities are only partially effective. In rats undergoing active and passive enhancement alone, heart allograft survival was increased to 25±12 days in two‐thirds, indefinitely in one‐third. After ART18 treatment, grafts survive 21±1 days. Grafts are accepted permanently in animals receiving full‐dose CsA (15 mg/kg x7), but are rejected acutely (c. 7 days) when subtherapeutic doses (1.5 mg/kg ×7) are used. However, when subtherapeutic doses of CsA are given in combination with immunological enhancement or with interleukin‐2‐receptor‐targeted therapy, graft survival increases dramatically, with permanent or markedly prolonged engraftment occurring in all instances. In the early phases of host unresponsiveness, both enhancement and IL‐2R‐targeted therapy spare selectively T cells with suppressor activity in vivo; in enhanced animals, the W3/25+ subset is responsible for prolonged graft survival, the OX8+ fraction is responsible in ART18‐treated animals and in CsA‐treated animals. Both subpopulations show suppressor activity in the later stages of combination treatment. IL‐3 production is increased significantly in these states of unresponsiveness, an observation also noted during maintenance CsA treatment; this seems to correlate with suppressor activity. Immunoperoxidase studies of the graft infiltrates emphasize the synergistic effects of combination treatments. Thus, subtherapeutic doses of CsA plus biologic host manipulations produce greatly increased graft survival by affecting selectively different host immune mechanisms.

Immune Complex-Type Deposits in the Fischer-344 to Lewis Rat Model of Renal Transplantation and a Subset of Human Transplant Glomerulopathy.

Background Antibody-mediated rejection is a leading cause for renal transplant loss. Rodent models are useful to dissect pathomechanisms and to develop treatment strategies. Although used for decades as a model, glomerular histopathological findings of Fischer-344 kidneys transplanted into Lewis rats have never been comprehensively described. Methods Kidneys from Fischer-344 rats were transplanted into Lewis rats as life-sustaining allografts without immunosuppression. Lewis isografts and normal Fischer-344 kidneys served as controls. Grafts were harvested at 9 days, 6 and 26 weeks. Histopathological examination included light microscopy, immunohistochemistry, and morphometry. Findings were compared with 51 human biopsies with transplant glomerulopathy. Results Most glomerular findings in rat allografts resembled human acute and chronic antibody-mediated rejection with glomerulitis, microthrombosis, microaneurysms, glomerular hypertrophy, podocyte loss, glomerular basement membrane splitting, and secondary focal and segmental glomerulosclerosis. In line with previous reports on nonendothelial antigens, glomerular immunoglobulin and C4d deposition was mostly nonendothelial. Only in 26-week allografts, we found mesangial and subendothelial immune complex-type electron-dense deposits. Similar deposits were found in 8 of 51 human biopsies with transplant glomerulopathy after rigorous exclusion of immune complexes of other cause, particularly recurrent glomerulonephritis and hepatitis C. Conclusions Thus, our model closely reflects the glomerular changes of acute antibody-mediated rejection in humans and of a special subset of human transplant glomerulopathy. The significance of alloimmune immune complex-type deposits in human transplants deserves further investigation.

Monocytic Tissue Transglutaminase in a Rat Model for Reversible Acute Rejection and Chronic Renal Allograft Injury

Acute rejection is a major risk factor for chronic allograft injury (CAI). Blood leukocytes interacting with allograft endothelial cells during acute rejection were suggested to contribute to the still enigmatic pathogenesis of CAI. We hypothesize that tissue transglutaminase (Tgm2), a multifunctional protein and established marker of M2 macrophages, is involved in acute and chronic graft rejection. We focus on leukocytes accumulating in blood vessels of rat renal allografts (Fischer-344 to Lewis), an established model for reversible acute rejection and CAI. Monocytes in graft blood vessels overexpress Tgm2 when acute rejection peaks on day 9 after transplantation. Concomitantly, caspase-3 is activated, suggesting that Tgm2 expression is linked to apoptosis. After resolution of acute rejection on day 42, leukocytic Tgm2 levels are lower and activated caspase-3 does not differ among isografts and allografts. Cystamine was applied for 4 weeks after transplantation to inhibit extracellular transglutaminase activity, which did, however, not reduce CAI in the long run. In conclusion, this is the first report on Tgm2 expression by monocytes in vivo. Tgm2 may be involved in leukocytic apoptosis and thus in reversion of acute rejection. However, our data do not support a role of extracellular transglutaminase activity as a factor triggering CAI during self-limiting acute rejection.

Expression of Acetylcholine Receptors by Experimental Rat Renal Allografts

Chronic allograft injury (CAI) is a major cause for renal allograft dysfunction and characterized by vasculopathies, tubular atrophy, and fibrosis. We demonstrated that numerous leukocytes interact with vascular endothelial cells of allografts and produce acetylcholine, which contributes to vascular remodeling. The cholinergic system might be a promising target for the development of novel therapies. However, neither the cellular mechanisms nor the acetylcholine receptors involved in CAI are known. Kidney transplantation was performed in the Lewis to Lewis and in the Fischer-334 to Lewis rat strain combination, which is an established experimental model for CAI. Expression of nicotinic and muscarinic acetylcholine receptors mRNA was quantified in renal tissue by real-time RT-PCR on days 9 and 42 after surgery. We detected CHRNA2–7, CHRNA10, CHRNB2, CHRNB4, and CHRM1–3 mRNA in normal kidneys and in renal transplants. In contrast, CHRNA9, CHRM4, and CHRM5 mRNA remained below the threshold of detection. In renal allografts, CHRNA3 and CHRNB4 mRNA expression were dramatically reduced compared to isografts. In conclusion, we demonstrated that most acetylcholine receptor subtypes are expressed by normal and transplanted kidneys. Allograft rejection downmodulates CHRNA3 and CHRNB4 mRNA. The role of different acetylcholine receptor subtypes in the development of CAI remains to be established.

Thoracotomy versus video-assisted thoracoscopic surgery (VATS) in stage III empyema—an analysis of 217 consecutive patients

BackgroundPleural empyema is an infectious disease of the chest cavity, with a high morbidity and mortality. According to the American Thoracic Society, pleural empyema gets graduated into three stages, with surgery being indicated in intermediate stage II and chronic stage III. Evidence for the feasibility of a minimally-invasive video-assisted thoracoscopic approach in stage III empyema for pulmonary decortication is still little.MethodsRetrospective single-center analysis of patients conducted to surgery for chronic stage III pleural empyema from 05/2002 to 04/2014 either by video-assisted thoracoscopic surgery (VATS, n = 110) or conventional open surgery by thoracotomy (n = 107). Multiple regression analysis and propensity score matching was used to evaluate the influence of operation technique (thoracotomy versus VATS) on the length of post-operative hospitalization.ResultsOperation time was longer in the thoracotomy-group (p = 0.0207). Conversion rate from VATS to open surgery by thoracotomy was 4.5%. Post-operative complication- (61 patients in thoracotomy- and 55 patients in VATS-group), recurrence- (3 patients in thoracotomy- and 5 in VATS-group) and mortality-rates (6.5% in thoracotomy- and 9.5% in VATS-group) did not differ between both groups; the length of (post-operative) stay at intensive care unit was longer in the VATS-group (p = 0.0023). Duration of chest tube drainage and prolonged air leak rate were similar among both groups, leading to a similar overall and post-operative length of hospital stay in both groups. Adjusted to clinically and statistically relevant confounders, multiple regression analysis showed an influence of the surgical technique on length of post-operative stay after pair matching of the patients (n = 84 in each group) by propensity score (B = − 0.179 for thoracotomy = 0 and VATS = 1, p = 0.032) leading to a reduction of 0.836 days after a VATS-approach compared to thoracotomy.ConclusionsVATS in late stage (III) pleural empyema is feasible and safe. The decrease in post-operative hospitalization demonstrated by adjusted multiple regression analysis may indicate the minimally-invasive approach being safe, more tolerable for patients, and more effective.

Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1β Release by Pulmonary Epithelial Cells

Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1β. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1β release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae.

Lung decortication in phase III pleural empyema by video-assisted thoracoscopic surgery (VATS)—results of a learning curve study

Background Pleural empyema (PE) is a devastating disease with a high morbidity and mortality. According to the American Thoracic Society it is graduated into three phases and surgery is indicated in intermediate phase II and organized phase III. In the latter, open decortication of the lung via thoracotomy is the gold standard whereas the evidence for feasibility and safety of a minimally-invasive video-assisted thoracoscopic approach is still poor. Methods Retrospective single-center analysis of patients undergoing surgery for phase III PE from 02/2011 to 03/2015 [n=138, including n=130 VATS approach (n=3 of them with bilateral disease) and n=8 open approach]. The learning curve was assessed by grouping those 127 patients with unilateral disease who underwent a video-assisted thoracoscopic approach into two groups: VATS-1 (03/2011 to 06/2012, n=43) and VATS-2 (06/2012 to 03/2015, n=84). Results ASA-scores (P=0.0279) and rate of pre-operative drainage therapy (P=0.0534) were higher in VATS-2 patients. Operating times were longer in VATS-1 (P=0.0308), intra-operative complication as well as conversion to open surgery rates did both not differ. Rates of post-operative vasoconstrictive therapy (P=0.0191) and prolonged mechanical ventilation (P=0.0560) were both higher in VATS-2, however, post-operative length of stay (LOS) at intensive care unit, overall post-operative LOS and post-operative complication rate were similar in both groups. Conclusions Video-assisted thoracoscopic surgery is feasible for evacuation and decortication in late phase III PE. A learning curve of approximately 40 cases is sufficient to gain procedure-specific surgical skills and thus reduce the operating times sufficiently.

Protective loop ileostomy increases the risk for prolonged postoperative paralytic ileus after open oncologic rectal resection

PurposePostoperative gut dysmotility is a physiologic and frequent temporary reaction after major abdominal surgery. If paralysis merges into a prolonged ileus state, it causes significant morbidity and subsequently worse outcome and discomfort for the patients. Pathophysiology of pathologic prolonged postoperative paralytic ileus remains multifactorial.MethodsWe present a retrospective single-center analysis of patients, who underwent a primary open oncologic anterior rectal resection with primary anastomosis with or without defunctioning loop ileostomy during a 43-month period of observation. Primary endpoint was the rate of prolonged postoperative paralytic ileus, defined by the intravenous administration of neostigmine. Confounders for regression analysis were assessed by univariate analysis and correlations between confounders were examined. Odds ratio for prolonged postoperative paralytic ileus in patients with defunctioning loop ileostomy was estimated by a logistic regression model.ResultsOf 101 patients (62 male), 62 (61.39%) received defunctioning loop ileostomy. In univariate analysis, male gender and patients with ileostomy showed more frequently prolonged paralysis by tendency (both p = 0.07). Logistic regression analysis proves the influence of a defunctioning ileostomy on the development of prolonged postoperative paralytic ileus after oncologic rectal resection (p = 0.047). Odds ratio for prolonged postoperative paralytic ileus in patients with ileostomy was 4.96 [95% CI 1.02–24.03].ConclusionsAlthough the construction of defunctioning loop ileostomies during rectal resection is a safe, uncomplicated surgical procedure, they can cause significant postoperative morbidity for the patients. High fluid and electrolyte loss are well-known complications, but herewith we raise the evidence for prolonged gut paralysis in patients with defunctioning loop ileostomy.

C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation

Blood levels of the acute phase reactant C-reactive protein (CRP) are frequently measured as a clinical marker for inflammation, but the biological functions of CRP are still controversial. CRP is a phosphocholine (PC)-binding pentraxin, mainly produced in the liver in response to elevated levels of interleukin-1β (IL-1β) and of the IL-1β-dependent cytokine IL-6. While both cytokines play important roles in host defense, excessive systemic IL-1β levels can cause life-threatening diseases such as trauma-associated systemic inflammation. We hypothesized that CRP acts as a negative feedback regulator of monocytic IL-1β maturation and secretion. Here, we demonstrate that CRP, in association with PC, efficiently reduces ATP-induced inflammasome activation and IL-1β release from human peripheral blood mononuclear leukocytes and monocytic U937 cells. Effective concentrations are in the range of marginally pathologic CRP levels (IC50 = 4.9 µg/ml). CRP elicits metabotropic functions at nicotinic acetylcholine (ACh) receptors (nAChRs) containing subunits α7, α9, and α10 and suppresses the function of ATP-sensitive P2X7 receptors in monocytic cells. Of note, CRP does not induce ion currents at conventional nAChRs, suggesting that CRP is a potent nicotinic agonist controlling innate immunity without entailing the risk of adverse effects in the nervous system. In a prospective study on multiple trauma patients, IL-1β plasma concentrations negatively correlated with preceding CRP levels, whereas inflammasome-independent cytokines IL-6, IL-18, and TNF-α positively correlated. In conclusion, PC-laden CRP is an unconventional nicotinic agonist that potently inhibits ATP-induced inflammasome activation and might protect against trauma-associated sterile inflammation.