Good manufacturing practice production of CD34+ progenitor-derived NK cells for adoptive immunotherapy in acute myeloid leukemia.

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34+ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27).

Understanding HIV infection for the design of a therapeutic vaccine. Part II: Vaccination strategies for HIV.

HIV infection leads to a gradual loss CD4(+) T lymphocytes comprising immune competence and progression to AIDS. Effective treatment with combined antiretroviral drugs (cART) decreases viral load below detectable levels but is not able to eliminate the virus from the body. The success of cART is frustrated by the requirement of expensive lifelong adherence, accumulating drug toxicities and chronic immune activation resulting in increased risk of several non-AIDS disorders, even when viral replication is suppressed. Therefore, there is a strong need for therapeutic strategies as an alternative to cART. Immunotherapy, or therapeutic vaccination, aims to increase existing immune responses against HIV or induce de novo immune responses. These immune responses should provide a functional cure by controlling viral replication and preventing disease progression in the absence of cART. The key difficulty in the development of an HIV vaccine is our ignorance of the immune responses that control of viral replication, and thus how these responses can be elicited and how they can be monitored. Part one of this review provides an extensive overview of the (patho-) physiology of HIV infection. It describes the structure and replication cycle of HIV, the epidemiology and pathogenesis of HIV infection and the innate and adaptive immune responses against HIV. Part two of this review discusses therapeutic options for HIV. Prevention modalities and antiretroviral therapy are briefly touched upon, after which an extensive overview on vaccination strategies for HIV is provided, including the choice of immunogens and delivery strategies.

Understanding HIV infection for the design of a therapeutic vaccine. Part I: Epidemiology and pathogenesis of HIV infection.

HIV infection leads to a gradual loss CD4+ T lymphocytes comprising immune competence and progression to AIDS. Effective treatment with combined antiretroviral drugs (cART) decreases viral load below detectable levels but is not able to eliminate the virus from the body. The success of cART is frustrated by the requirement of expensive life-long adherence, accumulating drug toxicities and chronic immune activation resulting in increased risk of several non-AIDS disorders, even when viral replication is suppressed. Therefore there is a strong need for therapeutic strategies as an alternative to cART. Immunotherapy, or therapeutic vaccination, aims to increase existing immune responses against HIV or induce de novo immune responses. These immune responses should provide a functional cure by controlling viral replication and preventing disease progression in the absence of cART. The key difficulty in the development of an HIV vaccine is our ignorance of the immune responses that control of viral replication, and thus how these responses can be elicited and how they can be monitored. Part one of this review provides an extensive overview of the (patho-) physiology of HIV infection. It describes the structure and replication cycle of HIV, the epidemiology and pathogenesis of HIV infection and the innate and adaptive immune responses against HIV. Part two of this review discusses therapeutic options for HIV. Prevention modalities and antiretroviral therapy are briefly touched upon, after which an extensive overview on vaccination strategies for HIV is provided, including the choice of immunogens and delivery strategies.

Sequence evolution and escape from specific immune pressure of an HIV-1 Rev epitope with extensive sequence similarity to human nucleolar protein 6.

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition.

Laparoscopic restorative proctocolectomy: a 10-year experience of an evolving technique.

AIM: Restorative proctocolectomy is the definitive procedure for ulcerative colitis. The potential benefits of a minimal invasive approach make it appropriate to consider this approach provided that there are no adverse effects. The aim of the present study was to report our experience of laparoscopic assisted and 'total' laparoscopic restorative proctocolectomy (LRPC) and to highlight the difficulties encountered and the functional results obtained. METHOD: Electronic data were prospectively collected from all patients who underwent laparoscopic restorative proctocolectomy (LRPC) from October 1999 to April 2010. RESULTS: Seventy-two (40 male) patients [median body mass index 24 (19-48) kg/m(2) ] underwent LRPC over 10 years. Three had cancer. Forty-two had undergone a previous colectomy (laparoscopic in 38). There were 40 W- and 32 J-pouch reconstructions; seven were single-port procedures. The median operation time was 210 (75-330) min. There were five (7%) conversions, one of which resulted in immediate pouch failure. The median time to full diet was 36 (4-168) h, with a median hospital stay of 7 (2-64) days. There were seven (10%) readmissions. Complications were immediate (3%), early (22%) and long term (11%). The incidence of failure (excision or indefinite diversion) was 2.7%. The stoma has been closed in 67 patients. Median frequency of defaecation was 4/24 h, with normal continence in 90% and the ability to defer during the day in 98%. There was no new case of impotence or dyspareunia. CONCLUSION: Laparoscopic restorative proctocolectomy is safe and gives good results when performed by an experienced laparoscopic surgeon.

Medium-term results of stapled transanal rectal resection (STARR) for obstructed defecation and symptomatic rectal-anal intussusception.

AIM: Stapled transanal rectal resection (STARR) is an increasingly accepted treatment for obstructed defaecation syndrome (ODS) associated with internal rectal prolapse (IRP) and rectocoele. The aim of this study is to evaluate the medium to long-term outcomes of STARR for ODS. METHOD: The intermediate-term results of STARR used over a 9-year period were reviewed from the analysis of a prospectively maintained database. Patients were followed for a median period of 98 (95% CI 85-112, range 5-386) weeks. RESULTS: Three hundred and forty-four (234 woman) patients of median age 54 (19-90) years underwent STARR over a 9-year period. Preoperative symptoms included pelvic pain (93%), incomplete evacuation (90%), urgency (74%), a sensation of obstruction (65%) and rectal digitation (27%). Thirteen had the solitary rectal ulcer syndrome. Of 326 patients with follow-up data, 249 (76%) were followed beyond 1 year and 149 (43%) beyond 2 years. The ODS score improved [14.6 ± 5.4 pre vs 1.6 ± 3.1 post (P < 0.0001)] as did the faecal incontinence (FI) score [3.5 ± 3.3 pre vs 0.4 ± 1.3 post (P < 0.0001)]. Fifteen (4.3%) patients reported deterioration in FI, and 11 (3.2%) experienced new onset minor incontinence. Urgency was 72% at 8 weeks, 20% at 16 weeks, 11.5% at 52 weeks and 5% at 1.5 years. None of the 29 patients followed beyond 4 years reported urgency. Urgency was unrelated to sex, age or preoperative ODS symptoms (Mantel-Cox log-rank). Recurrent symptoms of ODS occurred in 4.9%. Eighty-one per cent of patients were highly satisfied with STARR and would recommend or have it again. CONCLUSION: STARR was successful for the treatment of selected patients with ODS and IRP. Postoperative faecal urgency rapidly decreases with time. It is not possible to predict who will develop urgency.

Laparoendoscopic single-site surgery is feasible in complex colorectal resections and could enable day case colectomy.

BACKGROUND: Fast-track surgery accelerates recovery, reduces morbidity, and shortens hospital stay. However, the benefits of laparoscopic versus open surgery remain unproven within a fast-track program. Case reports of laparoendoscopic single-site (LESS) colectomies are appearing with claims of cosmetic advantage and decreased parietal trauma. This report describes the largest case series of LESS colorectal surgery and its effects on recovery. METHODS: In this series, 20 consecutive unselected patients underwent LESS colorectal surgery including right hemicolectomy (n = 3), extended right hemicolectomy, high anterior resection (n = 2), low anterior resection involving total mesorectal excision (TME; n = 3), ileocolic anastomosis (n = 2, including 1 redo surgery), colectomy and ileorectal anastomosis (n = 4, including 1 with ventral mesh rectopexy), panproctocolectomy (n = 2), proctocolectomy and ileoanal pouch (n = 2) and an abdominoperineal excision of rectum. Single-port conventional instrumentation and transversus abdominus plane (TAP) block analgesia were used. The indications included cancer (n = 8), Crohn's disease (n = 4), ulcerative colitis (n = 3) complicated diverticulosis (n = 2), and slow-transit constipation (n = 3). Eight of the patients had undergone previous surgery. RESULTS: Most of the cases (90%) were managed successfully using the LESS technique and conventional instrumentation. Two operations (10%) were converted to standard laparoscopy, due to insufficient theater time and an unstable port. The operative time ranged from 45 to 240 min (median, 110 min). A normal diet was tolerated within 6 h by 7 patients and in 12 to 16 h (overnight) by 11 patients. Complications included anastomotic bleed (n = 1), ileus (n = 2), acute renal failure secondary to hyperphosphatemia and hypocalcemia (n = 1), urine retention (n = 1), and wound infection (n = 1). The median hospital stay was 46 h (range, 7-384 h). Eight patients were discharged within 24 h. There was one readmission (5%). CONCLUSION: Laparoendoscopic single-site colorectal resection using conventional instrumentation is feasible and safe when performed by an experienced team. The LESS approach may have advantages in terms of minimal pain, cosmesis, lower costs, and faster recovery. A randomized trial is required to confirm whether LESS offers a true patient benefit over standard laparoscopic resection.

Single incision laparoscopic (SILS) restorative proctocolectomy with ileal pouch-anal anastomosis.

BACKGROUND: Restorative proctocolectomy with ileoanal pouch is the definitive procedure in ulcerative colitis. The potential benefits afforded by a single incision laparoscopic (SILS) approach make it appropriate to consider. METHODS: Electronic data were prospectively collected from all patients who underwent SILS restorative proctocolectomy (SILS-RPC) between June 2009 and June 2010. RESULTS: Ten consecutive patients (4 male), with median BMI = 22 (range = 20-28 kg/m(2)) underwent SILS-LRPC over a 1-year period. Three had undergone a previous emergency laparoscopic colectomy. A single-port device (Covidien SILS™ or Olympus TriPort™) was positioned at the site of the existing or proposed temporary ileostomy (2.5-cm incision). The colon and rectum were extracted through the SILS site (n = 8) or transanally following a mucosectomy (n = 2). A 20-cm J pouch was constructed extracorporeally and returned via the ileostomy site. Pouch-anal anastomosis was performed intracorporeally (n = 8) or hand-sutured (n = 2) and a diverting loop ileostomy was created at the SILS port site. The median operation time was 185 min (range = 100-381). There were no conversions or additional ports required. Median time to full diet was 36 h (range = 4-48 h) with a median hospital stay of 3 days (range = 2-8 days). There were no 30-day readmissions. Complications included surgical emphysema with temperature and a panic attack. Nine stomas have been closed. All patients have spontaneity of defecation, with a median pouch frequency of four per day, including once at night. All are fully continent and able to defer during the day. One reported a dry ejaculate for 10 weeks. CONCLUSION: SILS restorative proctocolectomy is safe with good early functional outcomes when performed by an experienced laparoscopic surgeon.

Randomized clinical trial of the influence of local subcutaneous infiltration vs subcutaneous and deep infiltration of local anaesthetic on pain after appendicectomy.

INTRODUCTION: Local anaesthesia is commonly used to provide pain relief after surgery. The aim of this randomized, blinded prospective trial was to evaluate whether local subcutaneous and deep infiltration of local anaesthetic provides better postoperative pain control than subcutaneous infiltration alone. METHOD: Eighty-four patients undergoing open appendicectomy for presumed acute appendicitis were randomly assigned into two groups. Group A received local infiltration of the skin prior to incision with bupivacaine whilst group B received half the bupivacaine infiltrated into the skin and the other half deep-to-external oblique to create a local nerve field blockade. Postoperative pain was assessed, on a scale of 1 to 10, at 1, 4, 8 and 24 h postextubation. RESULTS: At time intervals of 1, 4 and 8 h, there was no significant difference in pain score between group A and group B. At 24 h group B had significantly lower pain scores than group A (2.3 vs 3.4, P = 0.016). CONCLUSION: Both methods of administration of local anaesthetic produced consistently low pain scores in the first 24 h after appendicectomy. There may be additional benefit in a local nerve field blockade in combination with local anaesthetic skin infiltration.

Dictionary of interfaces in proteins (DIP). Data bank of complementary molecular surface patches.

Molecular surface areas of proteins are responsible for selective binding of ligands and protein-protein recognition, and are considered the basis for specific interactions between different parts of a protein. This basic principle leads us to study the interfaces within proteins as a learning set for intermolecular recognition processes of ligands like substrates, coenzymes, etc., and for prediction of contacts occurring during protein folding and association. For this purpose, we defined interfaces as pairs of matching molecular surface patches between neighboring secondary structural elements. All such interfaces from known protein structures were collected in a comprehensive data bank of interfaces in proteins (DIP). The up-to-date DIP contains interface files for 351 selected Brookhaven Protein Data Bank entries with a total of about 160,000 surface elements formed by 12,475 secondary structures. For special purposes, the inclusion of additional structures or selection of subgroups of proteins can be performed in an easy and straightforward manner. Atomic coordinates of the constituents of molecular surface patches are directly accessible as well as the corresponding contact distances from given atoms to their neighboring secondary structural elements. As a rule, independent of the type of secondary structure, the molecular surface patches of the secondary structural elements can be described as quite flat bodies with a length to width to depth ratio of about 3:2:1 for patches consisting of more than ten atoms. The relative orientation between two docking patches is strongly restricted, due to the narrow distribution of the distances between their centers of mass and of the angles between their normal lines, respectively. The existing retrieval system for the DIP allows selection (out of the set of molecular patches) according to different criteria, such as geometric features, atomic composition, type of secondary structure, contacts, etc. A fast, sequence-independent 3-D superposition procedure was developed for automatic searches for geometrically similar surface areas. Using this procedure, we found a large number of structurally similar interfaces of up to 30 atoms in completely unrelated protein structures.

Conservation of substructures in proteins: interfaces of secondary structural elements in proteasomal subunits.

It is observed that during divergent evolution of two proteins with a common phylogenetic origin, the structural similarity of their backbones is often preserved even when the sequence similarity between them decreases to a virtually undetectable level. Here we analyzed, whether the conservation of structure along evolution involves also the local atomic structures in the interfaces between secondary structural elements. We have used as study case one protein family, the proteasomal subunits, for which 17 crystal structures are known. These include 14 different subunits of Saccharomyces cerevisiae, 2 subunits of Thermoplasma acidophilum and one subunit of Escherichia coli. The structural core of the 17 proteasomal subunits has 23 secondary structural elements. Any two adjacent secondary structural elements form a molecular interface consisting of two molecular patches. We found 61 interfaces that occurred in all 17 subunits. The 3D shape of equivalent molecular patches from different proteasomal subunits were compared by superposition. Our results demonstrate that pairs of equivalent molecular patches show an RMSD which is lower than that of randomly chosen patches from unrelated proteins. This is true even when patch comparisons with identical residues were excluded from the analysis. Furthermore it is known that the sequential dissimilarity is correlated to the RMSD between the backbones of the members of protein families. The question arises whether this is also true for local atomic structures. The results show that the correlation of individual patch RMSD values and local sequence dissimilarities is low and has a wide range from 0 to 0.41, however, it is surprising that there is a good correlation between the average RMSD of all corresponding patches and the global sequence dissimilarity. This average patch RMSD correlates slightly stronger than the C(alpha)-trace RMSD to the global sequence dissimilarity.

Inverse sequence similarity in proteins and its relation to the three-dimensional fold.

Nowadays the most successful strategy for the prediction of the tertiary structure of proteins is the homology-based modelling using known structures. A real chance to predict the general fold of a protein arises only in cases with a sufficient sequence homology (e.g. 27% over 100 residues). In this analysis we examine the phenomenon of inverse sequence similarity (ISS) in proteins and its structural meaning. In sequence data bases we found a lot of examples for ISS up to 34% identity over 204 residues and a surprisingly large number of self-inverse protein sequences. By inspection of inverse similar sequence pairs with known tertiary structures we observe that inverse sequence alignments above the threshold indicating structural similarity generally do not imply comparable folds for both. From our analysis we conclude that the straightforward employment of ISS for protein structure prediction fails even above the known threshold for 'safe similarity'.

The possible role of selenium in antioxidation in marine waders; a preliminary study.

In a marine wader, the oystercatcher (Haematopus ostralegus), the activity of the selenium-dependent enzyme glutathione peroxidase (GSH-Px) in the red blood cells (RBC) was measured. The average activity, 97 +/- 19 units/g Hb, is within the range reported for mammals. No correlation was observed between the selenium concentration and GSH-Px activity in the erythrocytes and it is calculated that only a small percentage of the selenium present in the RBC is bound to the enzyme. Therefore, it is concluded that the high selenium concentrations in the avian red cells cannot be ascribed to GSH-Px. It is argued that a function of selenium in antioxidation is still possible. The positive relationship found between selenium and iron concentrations in the tissues of the oystercatcher directs to such a role. A pitfall in the measurement of GSH-Px activity is outlined.

Have high selenium concentrations in wading birds their origin in mercury?

The relationship between selenium and mercury in marine waders from the Wadden Sea (known to have high tissue selenium concentrations), was investigated in the framework of the possibility that high mercury concentrations may have induced parallel selenium accumulation to detoxify the mercury. The selenium and mercury concentrations are shown to be negatively correlated in both the liver and kidneys of these birds. In the tissues and red blood cells of oystercatchers, positive correlations between the two elements are found. The atom increment Se/Hg ratio in the pooled oystercatcher tissue and red cell data is 32:1. This ratio greatly exceeds the 1:1 ratio found when selenium is accumulated to detoxify mercury. Furthermore, breeding females are able to excrete mercury independently of selenium from the red blood cells, probably into the eggs; the whole egg mercury concentrations of the local breeding populations are low. From these results it is concluded that the high selenium concentrations in waders do not have their origin in elevated levels of mercury.

Selenium, mercury, arsenic and cadmium in the lifecycle of the dunlin, Calidris alpina, a migrant wader.

Migrant waders accumulate pollutants from their marine moulting and wintering grounds in Western Europe. In examining the possible consequences on the reproduction of the Dunlin (Calidris alpina), it was found that no adverse effects are to be expected from selenium, mercury, arsenic or cadmium. Though selenium is accumulated in very high concentrations in the kidney, after the bird's departure from the marine environment for the freshwater breeding areas, levels decline rapidly. Mercury exposure in Western Europe is not excessive, as shown by the relatively low levels in the summer plumage. Feather element concentrations proved to be a useful indicator of the different moulting and wintering areas used by the birds.

Selenium status in Charadriiformes. Tissue distribution and seasonal, geographical, and species variation.

The distribution of selenium in a marine wader, the Oystercatcher (Haematopus ostralegus) is given by the levels in 15 tissues and plasma. Red blood cells (RBC) contain the highest level (23 mg/kg dry wt) followed by liver, lung, and kidney (17-19 mg/kg). Most other tissues range from 3-10 mg/kg. The average kidney and liver concentrations of the Oystercatcher belong to the concentrations characteristic in birds. However, the Oystercatcher's tissue selenium concentrations are in general four- to fivefold mammalian levels, but in liver and lung, 11- to 13-fold and in the RBC, 12- to 33-fold. The selenium plasma and RBC levels of the Oystercatcher vary during the year from 280 to 410 micrograms/L and 13 to 30 mg/kg dry wt, respectively; the plasma concentrations are positively correlated with the RBC selenium concentrations. An overview of literature data shows that the selenium kidney and liver concentrations of birds do not vary with geographical latitude and size (length) of the birds. In species of the orders Charadriiformes and Procellariiformes, high selenium kidney, and to a lesser extent liver, concentrations may occur. A function of selenium in antioxidation is suggested.

The bioavailability of various selenium compounds to a marine wading bird.

The uptake of dietary selenium (about 3.5 mg/kg AF dry wt) as selenomethionine, selenocystine, selenite, selenate, and fish selenium in the plasma and red blood cells (RBC) of the oystercatcher has been investigated. The birds received the various selenium compounds subsequently, for at least 9 wk. After dietary supplementation of selenocystine, selenite, and selenate, plasma selenium was about 350 micrograms/L and RBC selenium 2.1 mg/kg dry wt. After supplementation of selenomethionine, the plasma concentration increased to 630 micrograms/L, and the RBC concentration to 4.1 mg/kg dry wt. When the fodder contained 3.1 mg/kg fish Se, an average plasma and RBC concentration of 415 micrograms/L and 14.4 mg/kg dry wt, respectively, was measured. The maximal increase of the selenium concentration in the plasma was attained at first sampling, 14 d after a change in dietary selenium (selenomethionine or fish Se); the uptake seemed to be a concentration-regulated process. RBC concentrations (Y in mg/kg drug wt) increased with time (X in d) according to Y = a - b e-cX. Fifty percent of the total increase was attained within 17 d, suggesting that diffusion into the RBC played a role. The selenium concentration in the plasma was positively correlated with the (fish)Se concentration in the fodder; the RBC concentration (60 d after the change in diet) was positively correlated with the plasma concentration. When the diet contained fish Se, the blood selenium concentrations of the captive birds were similar to the concentrations measured in field birds. Fish Se is a yet undetermined selenium compound. The present experiment showed that fish Se differed from selenomethionine, selenocystine, selenite, or selenate in uptake from the food and uptake in the RBC.

Confocal microscopy in microgravity research.

We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.

Atypical lipoma of the tongue.

We report an atypical lipoma arising in the tongue of a 43-year-old man who presented with an indolent dorsal lingual swelling. Atypical lipomas contain multivacoulated lipoblasts, which distinguishes them from benign lipomas. The superficial location in this case distinguishes this tumour from well-differentiated liposarcoma, which is biologically similar in lacking the propensity for metastasis. The superficial location of atypical lipoma allows a complete resection, which is often not possible for the deep-seated counterpart. Atypical lipoma and well-differentiated liposarcoma, if left in situ, may undergo transition to de-differentiated liposarcoma. Atypical lipoma should be completely excised with a cuff of normal tissue in order to prevent repeated local recurrence and the possibility of de-differentiation.