Combination of immunoglobulins and natural killer cells in the context of CMV and EBV infection.

Cytomegalovirus (CMV)-specific hyperimmunoglobulin (CMV-HIG) is used to treat and prevent CMV infection in immunocompromised patients, and anti-CD20 monoclonal antibody is successfully used in the treatment for post-transplant lymphoproliferative disease caused by Epstein-Barr virus (EBV). Two immunological approaches have been suggested to further improve the control of viral reproduction in patients with active disease: first, the use of monoclonal antibodies with specificity against viral epitopes and second, coadministration of cells with the capacity to promote antibody-dependent cell-mediated cytotoxicity. Here, we have evaluated the effectiveness of these strategies in vitro (alone and in combination) with neutralization and cytotoxicity assays. Our results indicate that monoclonal antibodies (in particular SM5-1) can be as effective as CMV-HIG in neutralizing-cell-free CMV. Moreover, our data indicate that antibody-mediated elimination (either by moAb or by HIG) of EBV-infected cells can be significantly enhanced by NK cells. Using human NK cells that have been purified, cultured and expanded under GMP conditions, we were able to demonstrate that the combination of NK cells and antibodies could represent a feasible and highly effective clinical approach to achieve control of EBV infections. Especially in leukopenic patients with low numbers of ADCC-promoting cells, the combination of adoptively transferred NK cells and antiviral antibodies offers a promising strategy that should be tested in clinical trials.

Antiviral function and efficacy of polyvalent immunoglobulin products against CMV isolates in different human cell lines.

Primary infection and reactivation of human cytomegalovirus (CMV) remain a major problem in immunocompromised patients, frequently resulting in a life threatening CMV disease. Intravenous polyvalent (hyper)-immunoglobulins (IVIG) can be administered for therapy and prophylaxis of CMV infections. However, only limited data about the efficacy and mechanism of action of IVIG products against viral infections in vitro are available so far. In this study, the effect of IVIG on CMV infection in vitro was investigated using isolates from CMV-infected patients as well as the laboratory strains AD169 and TB40. A qualitative and quantitative comparison of five different commercially available IVIG products in different human cell lines was performed concerning their ability (1) to neutralize cell-free virus, (2) to inhibit cell-to-cell spread and cell-associated transmission and (3) to influence CMV mRNA levels. All IVIG tested exhibited a high neutralization activity in epithelial and endothelial cell cultures (50% inhibition dose <0.1 mg/ml). However, qualitative differences between the products could be demonstrated in neutralization tests using human embryonal lung fibroblasts (HELF). The IVIG products also significantly differed in their ability to inhibit cell-to-cell spread within an CMV-infected HELF monolayer displaying inhibition rates that varied between 61 and 100%. No correlation between the ability to neutralize cell-free virus and to inhibit cell-to-cell spread could be observed. The incubation with IVIG influenced the amount of CMV immediate early and late mRNA, as indicated by a significant reduction in CMV mRNA in infected epithelial cells after incubation with IVIG in a dose-dependent manner. This study suggests different antiviral functions of polyvalent IVIG and confirms their potential to inhibit a CMV infection in vitro, with profound differences between the hereby used IVIG products.

Quantitative distinction of cisplatin-sensitive and -resistant mouse fibrosarcoma cells grown in multicell tumor spheroids.

As a suitable model to study the growth behavior and therapeutic response of drug-resistant and -sensitive cells in three-dimensional coculture we have established multicellular spheroids generated from both cisplatin-sensitive and -resistant cells of a murine fibrosarcoma cell line. A drug resistant clone was derived from the parent cisplatin-sensitive cells by intermittent drug exposure in vitro. As a prerequisite for analysis of differential growth and treatment response of spheroid subpopulations, two efficient methods to discriminate between the two morphologically indistinguishable subpopulations in mixed spheroids were established. In the cisplatin-resistant cell line chosen for the present study, resistance is mainly due to an increased cellular metallothionein content and is therefore associated with increased resistance to CdCl2. Exposure of colonies to high concentrations of CdCl2 thus allowed selective elimination of sensitive colonies. Permanent labeling of either resistant or sensitive cells was achieved by introduction of the Escherichia coli beta-galactosidase marker gene with a retroviral vector system. The transformation of an uncolored galactose derivative by this enzyme into an indigo stain allowed detection of cells carrying and expressing the marker gene. The marker gene and CdCl2 method led to identical results when used simultaneously to distinguish quantitatively between resistant and sensitive colonies grown from plated cells of untreated or irradiated mixed spheroids. The retroviral labeling method was also used successfully in the study of intact spheroids, showing that in 1:1 mixed spheroids, cisplatin-sensitive parent cells accumulate in the spheroid periphery, outgrowing resistant cells and displacing them into the metabolically restricted spheroid center. Only when sensitive and resistant cells are initially mixed at a ratio of 1:9 are the resulting spheroids composed of equal proportions of the 2 cell types throughout 10-20 days after spheroid initiation.

Oesophagostomum bifurcum-induced nodular pathology in a highly endemic area of Northern Ghana.

Human infection with Oesophagostomum bifurcum is rare globally, but focally endemic and common in Ghana and Togo. Two clinical presentations are identified: uni-nodular disease, which may be recognized as a 'Dapaong Tumour', and multi-nodular disease. Here, we describe the prevalence of O. bifurcum infection and the association with nodular pathology in northern Ghana. The study was performed in October 2002. Out of a well-defined population of approximately 18000, 928 subjects of all ages were randomly selected for parasitological and ultrasound examination. In stool cultures, 44% had detectable third-stage O. bifurcum larvae present. Females were more often infected than males (P<0.05). In 34% of the samples, nodules were detected along the colon wall, with the ascending and the transverse colon being the most affected regions. Significant correlations existed between the intensity of infection and the presence of nodules, both at the village and the individual level (P<0.001 for both). Patients with multi-nodular pathology had significantly higher larval counts than patients with uni-nodular pathology. The present data suggest that nodular pathology, and probably the severity of the disease, are directly related to intensity of the infection.

Expression of human heat-shock protein 70 antigens and gamma/delta T-cell receptor antigens in human central nervous tissue.

The human gamma delta T cell receptor is normally expressed on lymphoid tissue. Because expression of different molecules of the T-cell system has been described on human brain cells, we examined the expression of T-cell receptor gamma delta antigens with a panel of various anti-gamma/delta TCR mAbs using immunohistochemistry on different regions of frozen human postmortem tissue of five different brains. We found expression of gamma/delta TCR antigens on brain tissue in different regions of the brain, probably on neurons. Using mAbs against the 70-kd human heat-shock-protein (hsp 70), immunohistochemistry showed staining of microglia. A polymerase chain reaction analysis using a highly sensitive primer sequence against the constant region delta sequence supports the notion that the gamma/delta TCR is expressed in human brain; however, the sequence cannot be assigned to a specific tissue with this method. Both heat shock proteins and the gamma/delta TCR seem to be involved in autoimmune processes, and their expression on colocalizing structures in human CNS may play a role in triggering neuropsychiatric autoimmune disorders.

Intratracheal furosemide in infants after cardiac surgery: its effects on lung mechanics and urinary output, and its levels in plasma and tracheal aspirate.

OBJECTIVE: Recent studies have suggested direct pulmonary effects of furosemide in asthmatics and infants with bronchopulmonary dysplasia. We tested the hypothesis that intratracheally administered furosemide also increases respiratory compliance in children after cardiac surgery, and investigated whether furosemide has a topical and/or systemic action. STUDY DESIGN: Prospective study with intra-individual control. In twelve infants and toddlers (age: 10 +/- 8 months, weight: 6.9 +/- 3 kg) mechanically ventilated for compromised lung mechanics after cardiac surgery, 0.5 mg/kg furosemide was intratracheally administered to the lungs. Lung mechanics were serially assessed using a computerised system (Sensormedics 2600) during a 2 h control and 2 h intervention period. Urine output was measured by an indwelling bladder catheter and levels of furosemide were determined in blood and tracheal aspirates. RESULTS: Static compliance improved within 30 min in all patients, reached a maximum of 44 (20-85)% above baseline and remained improved throughout the study (p < 0.05). An immediate, short and significant diuretic effect of intratracheally applied furosemide was observed. Furosemide levels 1 h after intervention were 795 ng/ml in the blood and 431 micrograms/ml (i.e. 1000-fold higher) in the tracheal aspirate. Changes in compliance were correlated only to urine output values over the 2 h (r = 0.82, p = 0.044, n = 9) after furosemide administration. CONCLUSION: We conclude that intratracheally applied furosemide improves static compliance in infants and toddlers with compromised lung mechanics after cardiac surgery. We demonstrated that furosemide is absorbed from the lung and has a systemic effect within 15 min after its intratracheal instillation.

Evidence for a long-term effect of a single dose of praziquantel on Schistosoma mansoni-induced hepatosplenic lesions in northern Uganda.

Treatment with praziquantel reduces the prevalence and intensity of Schistosoma mansoni infection. However, reversibility of periportal fibrosis of the liver, which potentially leads to fatal complications, is not unequivocally substantiated. In the Nile District of Uganda, 460 patients were parasitologically (Kato-Katz method) and ultrasonographically examined during October 1991, October 1992, and May 1994. Treatment with praziquantel at a dosage of 40 mg per kilogram of body weight was given in October 1991 and October 1992 to 460 individuals (group A). Another 192 patients were seen during the baseline study in October 1991 and missed the follow-up in October 1992 but took part in the second follow-up in May 1994. Thus, they received praziquantel only once in October 1991 (group B) and had an interval of 2.7 years until the next investigation in May 1994. Periportal thickening (PT) of the liver was assessed by ultrasound at each time point. Praziquantel therapy reduced the prevalence of S. mansoni in group A from 84% in 1991 to 31% in 1992 and 30% in 1994. The respective intensities of infection (geometric means of egg output) were 81 eggs per gram (epg) of stool in 1991, 31 epg in 1992, and 30 epg in 1994. Periportal thickening was found in 46% of patients in 1991, 32% of patients in 1992, and 35% of patients in 1994. Reversibility of PT was influenced by age (markedly lower reversibility in individuals older than 30 years) and sex (women and girls responded less favorably than did men and boys). Surprisingly, no significant difference was detected between group A and group B with respect to reversibility of PT The outcome between the 2 groups did not differ significantly. This may indicate that a single dose of praziquantel (as given to group B) may have a longer lasting effect than previously thought, that is, more than 2.5 years.

Preoperative ECMO in congenital cyanotic heart disease using the AREC system.

BACKGROUND: In cyanotic congenital heart disease, oxygen delivery is impaired either by reduced pulmonary perfusion or by limited entry of oxygenated blood into the systemic circulation. Additional impairment of oxygen delivery (eg, in pulmonary hypertension) leads to hypoxic cerebral damage. Preoperative extracorporeal membrane oxygenation enables oxygenation in otherwise untreatable cases. METHODS: In 3 neonates suffering from cyanotic congenital heart disease (1 with tricuspid atresia and 2 with transposition of the great arteries) with arterial desaturation despite application of prostaglandins, balloon atrioseptostomy, and eventually inhaled nitric oxide during intermittent positive-pressure ventilation with an inspired oxygen fraction of 1, oxygenation could only be established by means of preoperative extracorporeal membrane oxygenation. We used a venovenous single-lumen cannula tidal-flow extracorporeal membrane oxygenation system described by Chevalier and associates that has previously been used for extracorporeal lung support. In this system, called AREC (assistence respiratoire extra-corporelle), alternating clamps and a nonocclusive roller pump were used. RESULTS: All 3 survived. CONCLUSIONS: We conclude that the AREC system enables sufficient preoperative oxygenation in patients with cyanotic congenital heart disease and hypoxia in spite of all conventional therapeutic means. This provides a stable preoperative condition for elective palliation or correction.

Intrathecal EBV antibodies are part of the polyspecific immune response in multiple sclerosis.

OBJECTIVE: One mechanism underlying the link between multiple sclerosis (MS) and Epstein-Barr virus (EBV) might be a direct CNS infection. Viral CNS infections cause elevated antibody indices (AIs). Elevated EBV AIs were found in MS; however, patients with MS frequently show a polyspecific intrathecal immune response with elevated antiviral AIs. To discriminate whether elevated EBV AIs indicate a virus-driven or a polyspecific intrathecal immune response, we determined the intrathecal fraction of anti-EBV antibodies. METHODS: The fraction of intrathecally synthesized EBV-specific immunoglobulin G (IgG) of the total intrathecally synthesized IgG (F(S) anti-EBV) was determined in 24 patients with a clinically isolated syndrome (CIS) or MS and 3 patients with cerebral posttransplantation lymphoproliferative disorder (PTLD), all of whom had elevated EBV AIs. F(S) anti-measles and AIs for measles, rubella, varicella zoster, and herpes simplex virus were measured as well. The prevalence of an elevated EBV AI was analyzed in another 36 patients with CIS. RESULTS: Median F(S) anti-EBV in patients with CIS/MS was low (0.65%) and did not differ from F(S) anti-measles (0.9%). Median F(S) anti-EBV was about 40-fold higher in patients with cerebral PTLD than in patients with CIS/MS. All 24 patients with CIS/MS with an elevated EBV AI had at least one further elevated antiviral AI. Only 2 of 36 (5.6%) patients with CIS showed an intrathecal synthesis of anti-EBV antibodies. CONCLUSIONS: Intrathecally produced anti-EBV antibodies are part of the polyspecific intrathecal immune response in CIS/MS and only rarely detectable in patients with CIS, both arguing against a direct CNS infection with EBV in patients with CIS/MS.

Deficiency of laryngeal collagen type II in an infant with respiratory problems.

A dysmorphic infant is described who presented with laryngeal collapse leading to intubation and respiratory problems that were assigned clinically to the Sussman syndrome. The baby had repeated episodes of respiratory distress necessitating assisted ventilation. At 6 months old, uvulopharyngopalatotomy was done to enlarge the supraglottic airway without any benefit. Surgical reduction of the tongue and cricoid splitting did not ameliorate the respiratory distress; repeated extubation attempts failed with the baby developing stridor, respiratory distress, and episodes of cardiac arrest. At 10 months old he developed seizures and computed tomography showed diffuse cerebral atrophy consisted with hypoxic-ischaemic damage. He died at 17 months old. Western blots using antibodies against collagen alpha 1 (II) showed an absence of collagen type II in laryngeal tissue, which may explain the laryngeal collapse and impaired respiratory functions.

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts.

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.

Quasistatic volume-pressure curve to predict the effects of positive end-expiratory pressure on lung mechanics and gas exchange in neonates ventilated for respiratory distress syndrome.

The shape of the volume-pressure (V/P) curve indicates alveolar collapse if it is convex to the pressure axis and indicates overdistension if it is concave. Positive end-expiratory pressure (PEEP) should either improve or decrease compliance and oxygenation in neonates ventilated for respiratory distress syndrome (RDS), depending on predominance of either alveolar collapse or overdistension. To test this hypothesis, we determined quasistatic V/P curves in 13 preterm neonates and characterized their shape by an alveolar distension index (ADI) at PEEP levels of 2, 4, and 6 cm H2O. We calculated the ADI dividing the V/P ratio at a low tidal volume by the V/P ratio at a high tidal volume. This ADI was then related to the effect of PEEP changes on respiratory compliance and alveolar to arterial oxygen tension difference (AaDO2). ADI was assumed to indicate alveolar collapse if less than 1 and overdistension if more than 1. An increased PEEP in neonates with alveolar collapse (ADI less than 1) decreased AaDO2 more (12 vs 10 mm Hg/cm PEEP, not significant) and decreased compliance less (3 vs 17%/cm PEEP; P < 0.05) than in those neonates with alveolar overdistension (ADI more than 1). Conversely, a decreased PEEP in neonates with alveolar overdistension increased compliance more (19 vs 5%; not significant) and increased AaDO2 less (7 vs 26 mm Hg; P < .01) than in those with alveolar collapse. AaDO2 and compliance changes after PEEP alterations were significantly correlated to the ADI before PEEP alterations (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

[Acute renal vein thrombosis in children. Early detection with duplex and color-coded Doppler ultrasound]. / Akute Nierenvenenthrombose bei Kindern. Früher Nachweis mit Duplex- und farbkodierter Dopplersonographie.

Two children with clinically suspected renal vein thrombosis were evaluated by duplex Doppler sonography and colour flow imaging. Both cases presented unspecific findings with an enlarged kidney and loss of cortico-medullary delineation on gray-scale ultrasound, but an unusual flow pattern with retrograde plateau-like frequency shifts during diastole. No venous signal could be obtained. By colour flow imaging only the main renal artery and its proximal branches could be visualised with a reverberating oscillation of blood flow. In addition, partial thrombosis of inferior vena cava in one patient and iliac vein thrombosis in the other could be demonstrated. Clinical improvement during fibrinolytic therapy in one case and nephrectomy in the other case confirmed the diagnosis.

[Allergen analysis of mugwort pollen (Artemisia vulgaris) using crossed radioimmunoelectrophoresis and radioallergosorbent test inhibition]. / Allergenanalyse von Beifusspollen (Artemisia vulgaris) mittels gekreuzter Radioimmunelektrophorese (CRIE) und RAST-Hemmung.

It could be identified 43 antigens of mugwort pollen (Artemisia vulgaris), 31 migrating anodically and 12 cathodically, by means of crossed immunoelectrophoresis (CIE). Eleven antigens bound human IgE-antibodies in crossed radioimmunoelectrophoresis (CRIE). Two major allergens could be found, allergens 23 and 40.- The allergenic activity of mugwort pollen extract was examined by means of RAST-inhibition. Specificity of this reaction could be determined by a control trial with grass pollen extract.

Structure and regulated expression of angiotensin-converting enzyme and the receptor for angiotensin II.

The renin-angiotensin system maintains a homeostasis of blood pressure and blood volume. One component of this system is angiotensin-converting enzyme (ACE). There are two isozymes of ACE. The protein produced by vascular endothelium is termed "somatic ACE" and is regulated as a function of the growth state of these cells in vitro. The second isozyme, "testis ACE," is only produced by developing spermatozoa. The two ACE isozymes are the result of two distinct promoter regions within the ACE gene. Angiotensin II binds to specific receptors on the surface of cells. We have isolated cDNA encoding the AT1 subtype of receptor. This subtype is responsible for the hemodynamic consequences of angiotensin.

The genomic organization of the rat AT1 angiotensin receptor.

The AT1 receptor subtype modulates all of the hemodynamic effects of the vasoactive peptide, angiotensin II. In this report, we investigate the genomic organization of this important receptor. A rat genomic library was screened with fragments from the 5' region of a previously cloned cDNA, pCa18b, encoding the rat AT1 receptor. Two lambda clones were isolated and the hybridizing restriction fragments were sequenced. Comparison of the genomic and cDNA sequences reveals that the rat AT1 receptor has three exons. Two of the exons encode 5' untranslated sequence while the third exon encompasses the entire coding region, a small portion of the 5' untranslated region and the entire 3' untranslated sequence. Further analysis of the genomic sequence 5' to the start site of pCa18b demonstrates typical sequence motifs found in many eukaryotic promoters including a TATA box, a cap site and a potential Sp1 binding site. Southern analysis of genomic DNA indicates that the AT1 receptor subtype represented by pCa18b is encoded by one gene within the rat genome.

Transmembrane neuregulins interact with LIM kinase 1, a cytoplasmic protein kinase implicated in development of visuospatial cognition.

The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-alpha did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand.

Therapeutic strategy in a 9-month-old child with pulmonary sling: need for bronchoscopic evaluation.

A 9-month-old child presented suffering from repeated severe infections of the lower respiratory tract. Bronchoscopy revealed a tracheal stenosis, suggestive of a vascular anomaly of the great arteries. A second significant stenosis of the left main stem bronchus was observed that was suspected to be due to a ligamentum arteriosum. Heart catheterization confirmed the diagnosis of an abnormal origin of the left pulmonary artery (pulmonary sling). Based on the bronchoscopic and angiographic findings a pulmonary ring was suspected. A dissection of the ligamentum arteriosum was performed. At readmission 4 weeks postoperatively the child was asymptomatic and the left main bronchus was patent.

[Therapy refractory arterial hypotension after heart operation]. / Therapieresistente arterielle Hypotonie nach Herzoperation.

Adrenalin insufficiency associated with adrenal hemorrhage, is a rare complication after cardiac surgery in neonates. A boy suffering from transposition of the great arteries, who had an arterial switch-operation on day three of his life, acquired a bilateral adrenal hemorrhage. Clinically the situation resembled a septic shock. Despite large doses of catecholamines, he continued to have severe arterial hypotension, anuria, and kyperkalemia. The clinical condition did not change, although sepsis specific therapy was initiated. Consequently adrenal insufficiency, as a possible postoperative complication, was considered and prednisolon, initially in a dose of 15 mg/kg/d, was administered. The clinical condition improved dramatically. The diagnosis could be confirmed by ultrasound examination and determination of cortisol and ACTH plasma levels. Adrenal insufficiency was only transitory, adrenal sonography on day 135 returned to normal. The surgical procedure on heart-lung bypass, the obligatory anticoagulation and the perioperative stress have to be considered as pathogenetic factors.

Expression of T cell receptor gamma delta antigens in human brain tissue.

The human gamma delta T cell receptor (TCR) is normally expressed on lymphoid tissue. Since expression of different molecules of the T cell system has been described for human brain cells, we examined the expression of CD4 and TCR gamma delta antigens with a panel of various anti-gamma delta TCR monoclonal antibodies (mAbs) and an anti-CD4 mAb using immunohistochemistry on different regions of frozen human postmortem tissue from five different brains. We could confirm the expression of CD4 antigens. gamma delta TCR expression on brain tissue was found in different regions of the brain by immunohistochemistry. Double staining with anti-gamma delta TCR and antineuronal enolase (NSE) mAbs showed that gamma delta TCR+ cells were not stained by anti-NSE, although they were sometimes located near neurons and showed dendritic forms; they are possibly tissue-resident gamma delta T cells, as described in the skin. Polymerase chain reaction analysis using a highly sensitive primer sequence against the constant-region delta sequence supports, combined with immunohistochemistry findings, the notion that the gamma delta TCR is expressed in human brain.