Severe mesenteric ischemia with multiple organ failure in a patient previously treated with a humanized monoclonal antibody against programmed death receptor-1 (pembrolizumab), a case of pembrolizumab associated catastrophic antiphospholipid syndrome?

Immune checkpoint inhibitors are used in the treatment of different types of tumors including melanoma and non-small cell lung carcinoma. The use of these inhibitors is associated with a broad spectrum of immune-related adverse effects. Here we report a case of a patient admitted to the intensive care unit with multiple organ failure due to catastrophic antiphospholipid syndrome following treatment with pembrolizumab, an immune checkpoint inhibitor, because of metastatic melanoma. The presented patient had multiple organ failure of lung, gastro-intestinal, renal, and the liver. Vascular thrombosis was confirmed by both imaging (pulmonary embolism on computed tomography-thorax) and histopathological examination of the intestines. In combination with the presence of IgA anti-cardiolipin antibodies and initially IgM anti-cardiolipin antibodies, catastrophic antiphospholipid syndrome was suspected. Despite treatment with plasmapheresis and corticosteroids, the patient died due to multiple organ failure. Catastrophic antiphospholipid syndrome is difficult to recognize and has high mortality rates despite supportive treatment. In this case report, discussion is provided regarding the possible immunological mechanism behind catastrophic antiphospholipid syndrome during or after treatment with immune checkpoint inhibitors. It is important to realize that in modern intensive care unit, more patients with immune-related adverse effects of the treatment with immune checkpoint inhibitors will be admitted, because of an increase in the number of patients treated with these checkpoint inhibitors. When these patients are admitted on the intensive care unit, multi-disciplinary consultation is important because of the difficulty of early recognition and optimal treatment of these possible lethal side effects.

Vasculotide, an angiopoietin-1 mimetic, reduces pulmonary vascular leakage and preserves microcirculatory perfusion during cardiopulmonary bypass in rats.

BACKGROUND: Cardiopulmonary bypass (CPB) during cardiac surgery impairs microcirculatory perfusion and is paralleled by vascular leakage. The endothelial angiopoietin/Tie2 system controls microvascular leakage. This study investigated whether targeting Tie2 with the angiopoietin-1 mimetic vasculotide reduces vascular leakage and preserves microcirculatory perfusion in a rat CPB model. METHODS: Rats were subjected to 75 min of CPB after treatment with vasculotide or phosphate buffered solution as control or underwent a sham procedure. Microcirculatory perfusion and leakage were assessed with intravital microscopy (n=10 per group) and Evans blue dye extravasation (n=13 per group), respectively. Angiopoietin-1, -2, and Tie2 protein and gene expression were determined in plasma, kidney, and lung. RESULTS: CPB immediately impaired microcirculatory perfusion [5 (4-8) vs 10 (7-12) vessels per recording, P=0.002] in untreated CPB rats compared with sham, which persisted after weaning from CPB. CPB increased circulating angiopoeietin-1, -2, and soluble Tie2 concentrations and reduced Tie2 messenger ribonucleic acid (mRNA) expression in kidney and lung. Moreover, CPB increased Evans blue dye leakage in kidney [12 (8-25) vs 7 (1-12) µg g-1, P=0.04] and lung [and 23 (13-60) vs 6 (4-16) µg g-1, P=0.001] compared with sham. Vasculotide treatment preserved microcirculatory perfusion during and after CPB. Moreover, vasculotide treatment reduced Evans blue dye extravasation in lung compared with CPB control [18 (6-28) µg g-1vs 23 (13-60) µg g-1, P=0.04], but not in kidney [10 (3-23) vs 12 (8-25) µg g-1, P=0.38]. Vasculotide did not affect circulating or mRNA expression of angiopoietin-1, -2, and Tie2 concentrations compared with untreated CPB controls. CONCLUSIONS: Treatment with the angiopoietin-1 mimetic vasculotide reduced pulmonary vascular leakage and preserved microcirculatory perfusion during CPB in a rat model.

Reduction of vascular leakage by imatinib is associated with preserved microcirculatory perfusion and reduced renal injury markers in a rat model of cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass during cardiac surgery leads to impaired microcirculatory perfusion. We hypothesized that vascular leakage is an important contributor to microcirculatory dysfunction. Imatinib, a tyrosine kinase inhibitor, has been shown to reduce vascular leakage in septic mice. We investigated whether prevention of vascular leakage using imatinib preserves microcirculatory perfusion and reduces organ injury markers in a rat model of cardiopulmonary bypass. METHODS: Male Wistar rats underwent cardiopulmonary bypass after treatment with imatinib or vehicle (n=8 per group). Cremaster muscle microcirculatory perfusion and quadriceps microvascular oxygen saturation were measured using intravital microscopy and reflectance spectroscopy. Evans Blue extravasation was determined in separate experiments. Organ injury markers were determined in plasma, intestine, kidney, and lungs. RESULTS: The onset of cardiopulmonary bypass decreased the number of perfused microvessels by 40% in the control group [9.4 (8.6-10.6) to 5.7 (4.8-6.2) per microscope field; P<0.001 vs baseline], whereas this reduction was not seen in the imatinib group. In the control group, the number of perfused capillaries remained low throughout the experiment, whilst perfusion remained normal after imatinib administration. Microvascular oxygen saturation was less impaired after imatinib treatment compared with controls. Imatinib reduced vascular leakage and decreased fluid resuscitation compared with control [3 (3-6) vs 12 ml (7-16); P=0.024]. Plasma neutrophil-gelatinase-associated-lipocalin concentrations were reduced by imatinib. CONCLUSIONS: Prevention of endothelial barrier dysfunction using imatinib preserved microcirculatory perfusion and oxygenation during and after cardiopulmonary bypass. Moreover, imatinib-induced protection of endothelial barrier integrity reduced fluid-resuscitation requirements and attenuated renal and pulmonary injury markers.

Incidence, timing and outcome of AKI in critically ill patients varies with the definition used and the addition of urine output criteria.

BACKGROUND: Acute kidney injury (AKI) is a serious complication of critical illness with both attributed morbidity and mortality at short-term and long-term. The incidence of AKI reported in critically ill patients varies substantially with the population evaluated and the definitions used. We aimed to assess which of the AKI definitions (RIFLE, AKIN or KDIGO) with or without urine output criteria recognizes AKI most frequently and quickest. Additionally, we conducted a review on the comparison of incidence proportions of varying AKI definitions in populations of critically ill patients. METHODS: We included all patients with index admissions to our intensive care unit (ICU) from January 1st, 2014 until June 11th, 2014 to determine the incidence and onset of AKI by RIFLE, AKIN and KDIGO during the first 7 days of ICU admission. We conducted a sensitive search using PubMed evaluating the comparison of RIFLE, AKIN and KDIGO in critically ill patients RESULTS: AKI incidence proportions were 15, 21 and 20% respectively using serum creatinine criteria of RIFLE, AKIN and KDIGO. Adding urine output criteria increased AKI incidence proportions to 35, 38 and 38% using RIFLE, AKIN and KDIGO definitions. Urine output criteria detected AKI in patients without AKI at ICU admission in a median of 13 h (IQR 7-22 h; using RIFLE definition) after admission compared to a median of 24 h using serum creatinine criteria (IQR24-48 h). In the literature a large heterogeneity exists in patients included, AKI definition used, reference or baseline serum creatinine used, and whether urine output in the staging of AKI is used. CONCLUSION: AKIN and KDIGO criteria detect more patients with AKI compared to RIFLE criteria. Addition of urine output criteria detect patients with AKI 11 h earlier than serum creatinine criteria and may double AKI incidences in critically ill patients. This could explain the large heterogeneity observed in literature.

Pro-inflammatory cytokines in cryptoglandular anal fistulas.

BACKGROUND: Sphincter-preserving procedures for the treatment of transsphincteric fistulas fail in at least one out of every three patients. It has been suggested that failure is due to ongoing disease in the remaining fistula tract. Cytokines play an important role in inflammation. At present, biologicals targeting cytokines are available. Therefore, detection and identification of cytokines in anal fistulas might have implications for future treatment modalities. The objective of the present study was to assess local production of a selected panel of cytokines in anal fistulas, including pro-inflammatory interleukin (IL)-1ß and tumor necrosis factor α (TNF-α). METHODS: Fistula tract tissue was obtained from 27 patients with a transsphincteric fistula of cryptoglandular origin who underwent flap repair, ligation of the intersphincteric fistula tract or a combination of both procedures. Patients with a rectovaginal fistula or a fistula due to Crohn's disease were excluded. Frozen tissue samples were sectioned and stained using advanced immuno-enzyme staining methods for detection of selected cytokines, IL-1ß, IL-8, IL-10, IL-12p40, IL-17A, IL-18, IL-36 and TNF-α. The presence and frequencies of cytokine-producing cells in samples were quantitated. RESULTS: The key finding was abundant expression of IL-1ß in 93 % of the anal fistulas. Frequencies of IL-1ß-producing cells were highest (>50 positive stained cells) in 7 % of the anal fistulas. Also, cytokines IL-8, IL-12p40 and TNF-α were present in respectively 70, 33 and 30 % of the anal fistulas. CONCLUSIONS: IL-1ß is expressed in the large majority of cryptoglandular anal fistulas, as well as several other pro-inflammatory cytokines.

Real-time PCR to distinguish livestock-associated (ST398) from non-livestock-associated (methicillin-resistant) Staphylococcus aureus.

BACKGROUND: The Netherlands is one of the most densely populated countries in the world, with extensive livestock of pigs. In 2005, the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) was a fact, with a relatively high MRSA colonisation among pig farmers. These MRSA isolates mostly belonged to sequence type 398 (ST398). Compared to hospital-associated MRSA (HA-MRSA), severe infections due to LA-MRSA and transmission between individuals are still relatively rare. Therefore, LA-MRSA may warrant less stringent containment measures than HA-MRSA in hospital settings. RESULTS: The aim of this study was to develop a rapid diagnostic tool to distinguish LA-MRSA from non-LA-MRSA in aid of infection control. Here, we show that ST398 strains can be readily detected with real-time polymerase chain reaction (PCR). Analysis of a large panel of related and unrelated microorganisms confirmed that the real-time ST398 PCR (ST398-qPCR) assay does not cross-react with other microorganisms or with non-LA-S. aureus strains. ST398-qPCR analysis of MRSA isolates collected in 2010, 2011 and 2012 at the Jeroen Bosch Hospital (n = 275) showed that an average of 78 % of MRSA belonged to sequence type ST398. CONCLUSION: We conclude that the ST398 real-time PCR is a reliable assay to detect LA-S. aureus and anticipate that the use of this assay can prevent the unnecessary closing of hospital wards, which may lead to substantial savings for the health care system.

Decreased systemic IL-7 and soluble IL-7Rα in multiple sclerosis patients.

Polymorphisms (single-nucleotide polymorphism (SNP)) in the interleukin-7 receptor-α (IL-7Rα)/IL-7 pathway are associated with an increased risk to develop multiple sclerosis (MS). The rs6897932 SNP in the IL-7Rα leads to increased soluble IL-7Rα production. Given the functional interaction between sIL-7Rα, membrane-bound IL-7Rα and IL-7, we assessed IL-7, mIL-7Rα and sIL-7Rα levels in MS patients and healthy controls (HCs). One-hundred and twenty eight MS patients had significantly lower sIL-7Rα levels compared with 73 HCs. The levels of sIL-7Rα increased dose-dependent upon rs6897932 [C] risk allele carriership in both HCs and MS. Next, we hypothesized that lower sIL-7Rα could result in a higher mIL-7Rα to soluble IL-7Rα ratio. Indeed, 52 MS patients had significantly increased mIL-7Rα to sIL-7Rα ratio for both CD4 and CD8 T cells compared with 44 HCs. Given the supposed role of IL-7 in autoimmunity, we determined whether sIL-7Rα influences IL-7 levels. IL-7 levels were significantly decreased in 40 MS patients compared with 40 HCs. In conclusion, MS patients had lower free IL-7 and a higher membrane to soluble IL-7Rα ratio. The soluble IL-7Rα levels correlate with the rs6897932 [C] risk allele carriership. The skew at the IL-7 and IL-7Rα level may influence responsiveness of IL-7Rα(+) cells.

MicroRNA-126 contributes to renal microvascular heterogeneity of VCAM-1 protein expression in acute inflammation.

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

Orbit-infiltrating mast cells, monocytes, and macrophages produce PDGF isoforms that orchestrate orbital fibroblast activation in Graves' ophthalmopathy.

PURPOSE: Platelet-derived growth factors (PDGF) are regulators of fibroblast activity that may be involved in the pathophysiology of Graves' ophthalmopathy (GO). We unraveled the expression and origin of PDGF family members in GO orbital tissue and investigated the effect of PDGF isoforms on IL-6 and hyaluronan production and proliferation by orbital fibroblasts. METHODS: PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGF-Rα, and PDGF-Rß expression was determined by real-time quantitative PCR and PDGF-A and PDGF-B protein expression was determined by Western blot in orbital tissues. Orbital tissues were immunohistochemically stained for PDGF-A and PDGF-B expression, together with stainings for T cells, monocytes, B cells, macrophages, and mast cells. Effects of PDGF-AA, PDGF-AB, and PDGF-BB on orbital fibroblast proliferation and IL-6 and hyaluronan production were examined. Finally, effects of PDGF-BB- and PDGF-AA-neutralizing antibodies on IL-6 and hyaluronan production in GO whole orbital tissue cultures were tested. RESULTS: GO orbital tissue showed increased PDGF-A and PDGF-B mRNA and protein levels. Increased numbers of PDGF-A- and PDGF-B-positive monocytes, macrophages, and mast cells were present in GO orbital tissue. PDGF-BB stimulated proliferation and hyaluronan and IL-6 production by orbital fibroblasts the most, followed by PDGF-AB and PDGF-AA. Finally, in particular imatinib mesylate and PDGF-BB-neutralizing antibodies reduced IL-6 and hyaluronan production by whole orbital tissue cultures from GO patients. CONCLUSIONS: In GO, mast cells, monocytes, and macrophages may activate orbital fibroblasts via secretion of especially PDGF-AB and PDGF-BB. Preclinical studies with whole orbital tissue cultures show that blocking PDGF-B chain containing isoforms can be a promising treatment for GO.

Serum leptin levels during pregnancy in multiple sclerosis.

BACKGROUND: Disease activity in patients with multiple sclerosis (MS) is suppressed during pregnancy, whereas attack frequency increases after delivery. It is yet unclear, which immuno - endocrinological processes mediate these disease fluctuations. Leptin has been identified as a hormone that can influence inflammatory activity. OBJECTIVE: The aim of this study was to investigate whether pregnancy-induced fluctuations of serum leptin levels differed between patients with MS and controls and whether serum leptin levels correlate with periods of enhanced and diminished disease activity. METHODS: Women with MS and healthy women were prospectively followed during and after pregnancy. The MS group could be studied already at a timepoint before pregnancy. Serum leptin and soluble leptin receptor (SLR) levels were measured using enzyme-linked immunosorbent assay. RESULTS: Pre-pregnancy serum leptin levels were (mean +/- SD) 22.9 +/- 12.8 ng/ml in the MS group. These levels increased in the third trimester to 28.5 +/- 15.0 ng/ml (P = 0.007). The third trimester serum leptin levels in healthy women were comparable, 29.4 +/- 19.0 ng/ml. Serum leptin levels after delivery dropped to 18.5 +/- 12.8 ng/ml in women with MS (P < 0.001) and to a lesser extend (22.0 +/- 17.5 ng/ml) in healthy women (P = 0.04). SLR levels showed the same pattern. Remarkably, women with the highest relative decrease in serum leptin levels after delivery had more often a postpartum relapse (P = 0.008). CONCLUSION: In women with MS, leptin increased during late pregnancy. A postdelivery drop in leptin levels was observed in both the MS and control group. The postdelivery drop was associated with the occurrence of postpartum relapse.

Novel monoclonal antibodies against proteolipid protein peptide 139-151 demonstrate demyelination and myelin uptake by macrophages in MS and marmoset EAE lesions.

Experimental autoimmune encephalomyelitis (EAE) induced by immunization of mice with epitopes of the proteolipid protein (PLP), a major myelin constituent, forms a useful model for the study of multiple sclerosis (MS). In addition, MS patients display PLP-specific T- and B-cell responses, suggesting that PLP reactivity is relevant to pathogenesis.Here, the generation and characterization of a panel of mouse monoclonal antibodies (Mab) against PLP139-151, the prominent encephalitogenic sequence in SJL/J mice is described. Five Mab were generated by conventional immunization of an SJL/J mouse and hybridoma generation. These Mab reacted well with the PLP139-151 peptide in ELISA and belonged to the IgG2a and IgG2b subclasses, consistent with CD4+ T helper 1-cell-supported antibody formation. The Mab also efficiently detected PLP peptide-BSA conjugates in Western blot, confirming their multi-assay applicability. The Mab were subsequently used to determine the occurrence of demyelination in brains of MS patients and marmoset monkeys with EAE. Immunohistochemistry on both paraffin and frozen sections demonstrated a homogeneous expression of PLP139-151 in normal myelin, and a complete absence in lesions containing demyelinated areas, confirming that the Mab can be used as a general myelin marker. In active demyelinating MS lesions, the Mab visualized the peptide in the cytoplasm of macrophages containing phagocytosed myelin. In conclusion, this panel of Mab against the encephalitogenic PLP139-151 epitope forms a useful tool for further study of autoantigen expression, demyelination/remyelination and the staging of lesional activity in MS patients, as well as in EAE models in distinct animal species.

Prevention of experimental autoimmune encephalomyelitis in the common marmoset (Callithrix jacchus) using a chimeric antagonist monoclonal antibody against human CD40 is associated with altered B cell responses.

Inhibition of CD40-CD40 ligand interaction is a potentially effective approach for treatment of autoimmune diseases, such as multiple sclerosis. We have investigated this concept with a chimeric antagonist anti-human CD40 mAb (ch5D12) in the marmoset monkey experimental autoimmune encephalomyelitis (EAE) model. Marmosets were immunized with recombinant human myelin oligodendrocyte glycoprotein (rMOG) and treated from the day before immunization (day -1) until day 50 with either ch5D12 (5 mg/kg every 2-4 days) or placebo. On day 41 after the induction of EAE, four of four placebo-treated monkeys had developed severe clinical EAE, whereas all animals from the ch5D12-treated group were completely free of disease symptoms. High serum levels of ch5D12 associated with complete coating of CD40 on circulating B cells were found. At necropsy placebo- and ch5D12-treated animals showed similar MOG-specific lymphoproliferative responses in vitro, but ch5D12 treatment resulted in strongly reduced anti-MOG IgM Ab responses and delayed anti-MOG IgG responses. Most importantly, treatment with ch5D12 prevented intramolecular spreading of epitope recognition. Postmortem magnetic resonance imaging and immunohistologic analysis of the CNS showed a markedly reduced lesion load after ch5D12 treatment. In conclusion, the strong reduction of clinical, pathological, and radiological aspects of EAE by ch5D12 treatment in this preclinical model points to a therapeutic potential of this engineered antagonist anti-CD40 mAb for multiple sclerosis.

Carrier-mediated enhancement of cognate T cell help: the basis for enhanced immunogenicity of meningococcal outer membrane protein polysaccharide conjugate vaccine.

Haemophilus influenzae type b capsular polysaccharide (PRP) conjugate vaccines, which are thought to induce T cell-dependent antibody production, induce protective responses after a single dose in individuals under 15 months of age. However, multiple doses of these vaccines are required to induce protective antibody responses in infants, with the exception of PRP conjugated to meningococcal outer membrane proteins (OMPC), which does so after a single dose. The basis for this difference is not fully understood, although others have proposed that OMPC and porins, the major protein component of OMPC, act as adjuvants or mitogens. In this report OMPC is shown to enhance CD40 ligand-mediated, T cell-dependent antibody production in mice. This paralleled the induction by OMPC of CD86, CD80 and CD40 costimulatory molecules on human neonatal and murine B cells and of Th1 cytokines. Neither porins nor lipopolysaccharide fully reproduced the effects of OMPC. These studies indicate that OMPC acts both as carrier and adjuvant, and thereby enhances T cell-dependent antibody responses in human infants.

Bacterial peptidoglycan and immune reactivity in the central nervous system in multiple sclerosis.

Multiple sclerosis is believed to result from a CD4+ T-cell response against myelin antigens. Peptidoglycan, a major component of the Gram-positive bacterial cell wall, is a functional lipopolysaccharide analogue with potent proinflammatory properties and is conceivably a mediator of sterile inflammation. Here we demonstrate that peptidoglycan is present within antigen-presenting cells in the brain of multiple sclerosis patients. These cells have macrophage and dendritic cell characteristics, and are immunocompetent as evidenced by co-expression of inflammatory cytokines and co-stimulatory molecules. In addition, intrathecal plasma cells specific for peptidoglycan are present in multiple sclerosis brain tissue, and antibodies binding peptidoglycan are present in CSF during active disease. Peptidoglycan may thus contribute to T- and B-cell activity during brain inflammation without a requirement for local bacterial replication.

A new primate model for multiple sclerosis in the common marmoset.

Experimental autoimmune encephalomyelitis (EAE) in outbred marmoset monkeys (Callithrix jacchus) is a recently developed nonhuman primate model of multiple sclerosis. Here, Bert 't Hart and colleagues compare this model to EAE in rhesus monkeys, highlighting autoimmune mechanisms in CNS inflammation and demyelination, including the role of major histocompatibility complex restriction and preclinical evaluation of innovative immunotherapies.

Pararosaniline fixation for detection of co-stimulatory molecules, cytokines, and specific antibody.

Integral immunohistochemical analysis of immune responses in frozen sections requires that, in addition to constitutively expressed membrane CD markers, less stable determinants can be reliably visualized. Therefore, we compared the commonly used acetone fixation method with pararosaniline fixation for six determinant categories. These categories included selected constitutively expressed markers, inducible co-stimulatory molecules, pro- and anti-inflammatory cytokines (including the novel cytokine IL-18, also known as IGIF and IL-1gamma), antigen-specific antibody in plasma cells, bacterial peptidoglycan, and lysosomal acid phosphatase activity. Human spleen and mouse spleen activated by agonistic anti-CD40 antibody or TNP-Ficoll immunization were analyzed in parallel with brain tissue from multiple sclerosis (MS) patients and marmoset monkeys with experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Fixation with pararosaniline resulted in better morphology of all tissues and inhibited endogenous alkaline phosphatase activity in brain tissue. Most determinants could be reliably detected. Staining sensitivity and intensity were markedly increased for selected determinant-tissue combinations, e.g., for IL-4 in human spleen and CD40 in human and mouse spleen. These data show that pararosaniline is a useful alternative to acetone, resulting in superior morphology and specific staining for selected determinant-tissue combinations. This provides additional flexibility for in situ analysis of immune reactivity.

Differential In situ cytokine profiles of Langerhans-like cells and T cells in Langerhans cell histiocytosis: abundant expression of cytokines relevant to disease and treatment.

The pathogenesis of Langerhans cell histiocytosis (LCH) remains poorly understood. To further elucidate LCH pathogenesis, we analyzed the expression of 10 cytokines relevant to cellular recruitment and activation at the protein level in 14 patients and identified the lesional cells responsible for cytokine production in situ by immunohistochemistry. The cytokines investigated included the hematopoietic growth factors interleukin-3 (IL-3), IL-7, and granulocyte-macrophage colony-stimulating factor (GM-CSF); the lymphocyte regulatory cytokines IL-2, IL-4, and IL-10; the inflammatory regulators IL-1alpha and tumor necrosis factor-alpha (TNF-alpha); and the effector cell-activating cytokines IL-5 and interferon-gamma (IFN-gamma). In all specimens, CD1a(+) histiocytes (LCH cells) and CD3(+) T cells produced large amounts of cytokines, creating a true cytokine storm. IL-2, IL-4, IL-5, and TNF-alpha were produced exclusively by T cells, whereas only IL-1alpha was produced by LCH cells. Equal numbers of LCH cells, T cells, and macrophages produced GM-CSF and IFN-gamma. Equal numbers of LCH cells and macrophages produced IL-10, whereas IL-3 was produced by T cells and macrophages. IL-7 was only produced by macrophages. Eosinophils, present in some specimens, were partially responsible for the production of IL-5, IFN-gamma, GM-CSF, IL-10, IL-3, and IL-7. Expression of all cytokines, abundant in most biopsies, was irrespective of age, gender, or site of biopsy. These findings emphasize the role of T cells in LCH. The juxtaposition of T cells and LCH cells suggests that both cells interact in a cytokine amplification cascade, resulting from stimulation of autocrine and paracrine stimulatory loops. This cascade can be linked directly to the development of LCH through recruitment, maturation, and proliferation of LCH cells. The cytokines studied are known to be involved in the development of other characteristic features of LCH, such as fibrosis, necrosis, and osteolysis.

Localization in situ of costimulatory molecules and cytokines in B-cell non-Hodgkin's lymphoma.

Costimulatory molecules are essential in cognate interactions between T and B lymphocytes. To study the prerequisites of functional interactions between malignant B cells and intermingled T cells in B-cell non-Hodgkin's lymphomas (B-NHL), we examined the expression of CD40, CD80 and CD86 and their ligands CD40 ligand (CD40L, CD154), CD28 and CTLA4 (CD152) using immunohistochemistry and confocal laser scanning microscopy. Almost all mucosa-associated lymphoid tissue (MALT) NHL were positive for CD40 and CD80 and in nine out of 14 cases were positive for CD86. The majority of follicle centre cell lymphomas (FCCL) expressed CD40, but were heterogeneous in their expression of CD80 and CD86. Most diffuse large cell lymphomas (DLCL) were CD80+, but lacked expression of CD86. These patterns reflect the differences in phenotype of normal marginal-zone B cells (as counterparts of MALT NHL) and germinal centre cells (as counterparts of FCCL and DLCL). Counter-receptors on T cells were detectable in 13 of 14 MALT NHL, 12 of 16 FCCL but only occasionally in DLCL (three of 12 cases). A subgroup of FCCL was identified with T-cell expression of CD40L, CD28 and CTLA4 simultaneously with strong expression of CD40 and CD86 on the tumour B cells. These results indicate that MALT NHL and a subset of FCCL are most optimally equipped for functional interactions with T cells. This may be supported by the demonstration of cytokine production - mainly in T cells - in MALT NHL [interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-10] and FCCL (IL-2, IFN-gamma) and to a lesser extent in DLCL.

Therapy with antibodies against CD40L (CD154) and CD44-variant isoforms reduces experimental autoimmune encephalomyelitis induced by a proteolipid protein peptide.

Interactions between mononuclear cells are required for the formation of inflammatory infiltrates in the CNS and the activation of cellular effector functions provoking demyelination in MS. Membrane-expressed costimulatory molecules are crucial to such interactions. We therefore investigated whether two costimulatory molecules, CD40L (CD154, expressed on activated CD4-possible T cells) and selected CD44-variant isoforms (expressed on activated CD4-positive T cells), are targets for immunotherapy in MS. The model of experimental autoimmune encephalomyelitis (EAE) induced in SJL-mice by immunization with a peptide derived from the proteolipid protein (PLP139-151) was optimized to address these questions. A previous observation that anti-CD40L (CD154) monoclonal antibodies can effectively prevent EAE in this model was confirmed, and extended by demonstrating that CD40 is expressed by cells of the monocytic lineage infiltrating the spinal cord. In vivo treatment with antibody against the standard isoform of CD44 (CD44s or CD44H) did not affect disease burden. In contrast, combined treatment with antibodies against the isoforms CD44v6, v7 and v10, which are thought to be involved in inflammatory processes, reduced the disease burden considerably. In addition, CD44v10-expressing cells were detected in the spinal cord. These data support the idea that CD40-CD40L interactions form a target for immunotherapy of MS, and indicate that cells expressing CD44v6, v7 and/or v10-containing isoforms have such potential as well.

Expression of accessory molecules and cytokines in acute EAE in marmoset monkeys (Callithrix jacchus).

Accessory molecules and cytokines are involved in the immunopathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) in rodent models, and are potential targets for immunotherapy. Evaluation of such experimental therapies requires appropriate animal models. Therefore, we analysed the expression of selected accessory molecules and cytokines in the brain of marmoset monkeys (Callithrix jacchus) with acute EAE, a newly described non-human primate model for MS. All animals experienced active disease clinically and histopathologically with strong resemblance to MS. Perivascular infiltrates of mononuclear cells showed abundant expression of CD40. CD40 was expressed on macrophages, indicating that T cell priming and macrophage effector functions may result from local CD40-CD40L interactions. CD40 ligand (CD40L) and B7-2 (CD86) were also expressed, but to a lower extent, while B7-1 (CD80) expression was limited. Both pro-inflammatory and anti-inflammatory cytokines were produced within individual lesions during active disease (IFN-alpha, IFN-gamma, TNF-alpha, IL-1alpha, IL-1beta, IL-2, IL-4, IL-10 and IL-12). This suggests that relative levels rather than sequential expression of Th1- and Th2-type cytokines determine disease activity. These findings demonstrate the value of EAE in marmoset monkeys as a model to assess the role of accessory molecules and cytokines in multiple sclerosis, and to evaluate targeted intervention.