Visible and near-infrared bulk optical properties of raw milk.

The implementation of optical sensor technology to monitor the milk quality on dairy farms and milk processing plants would support the early detection of altering production processes. Basic visible and near-infrared spectroscopy is already widely used to measure the composition of agricultural and food products. However, to obtain maximal performance, the design of such optical sensors should be optimized with regard to the optical properties of the samples to be measured. Therefore, the aim of this study was to determine the visible and near-infrared bulk absorption coefficient, bulk scattering coefficient, and scattering anisotropy spectra for a diverse set of raw milk samples originating from individual cow milkings, representing the milk variability present on dairy farms. Accordingly, this database of bulk optical properties can be used in future simulation studies to efficiently optimize and validate the design of an optical milk quality sensor. In a next step of the current study, the relation between the obtained bulk optical properties and milk quality properties was analyzed in detail. The bulk absorption coefficient spectra were found to mainly contain information on the water, fat, and casein content, whereas the bulk scattering coefficient spectra were found to be primarily influenced by the quantity and the size of the fat globules. Moreover, a strong positive correlation (r ≥ 0.975) was found between the fat content in raw milk and the measured bulk scattering coefficients in the 1,300 to 1,400 nm wavelength range. Relative to the bulk scattering coefficient, the variability on the scattering anisotropy factor was found to be limited. This is because the milk scattering anisotropy is nearly independent of the fat globule and casein micelle quantity, while it is mainly determined by the size of the fat globules. As this study shows high correlations between the sample's bulk optical properties and the milk composition and fat globule size, a sensor that allows for robust separation between the absorption and scattering properties would enable accurate prediction of the raw milk quality parameters.

Alignment to natural and imposed mismatches between the senses.

Does the nervous system continuously realign the senses so that objects are seen and felt in the same place? Conflicting answers to this question have been given. Research imposing a sensory mismatch has provided evidence that the nervous system realigns the senses to reduce the mismatch. Other studies have shown that when subjects point with the unseen hand to visual targets, their end points show visual-proprioceptive biases that do not disappear after episodes of visual feedback. These biases are indicative of intersensory mismatches that the nervous system does not align for. Here, we directly compare how the nervous system deals with natural and imposed mismatches. Subjects moved a hand-held cube to virtual cubes appearing at pseudorandom locations in three-dimensional space. We alternated blocks in which subjects moved without visual feedback of the hand with feedback blocks in which we rendered a cube representing the hand-held cube. In feedback blocks, we rotated the visual feedback by 5° relative to the subject's head, creating an imposed mismatch between vision and proprioception on top of any natural mismatches. Realignment occurred quickly but was incomplete. We found more realignment to imposed mismatches than to natural mismatches. We propose that this difference is related to the way in which the visual information changed when subjects entered the experiment: the imposed mismatches were different from the mismatch in daily life, so alignment started from scratch, whereas the natural mismatches were not imposed by the experimenter, so subjects are likely to have entered the experiment partly aligned.

A randomised controlled trial of spinal manipulative therapy in acute low back pain.

OBJECTIVE: To determine whether treatment with spinal manipulative therapy (SMT) administered in addition to standard care is associated with clinically relevant early reductions in pain and analgesic consumption. METHODS: 104 patients with acute low back pain were randomly assigned to SMT in addition to standard care (n = 52) or standard care alone (n = 52). Standard care consisted of general advice and paracetamol, diclofenac or dihydrocodeine as required. Other analgesic drugs or non-pharmacological treatments were not allowed. Primary outcomes were pain intensity assessed on the 11-point box scale (BS-11) and analgesic use based on diclofenac equivalence doses during days 1-14. An extended follow-up was performed at 6 months. RESULTS: Pain reductions were similar in experimental and control groups, with the lower limit of the 95% CI excluding a relevant benefit of SMT (difference 0.5 on the BS-11, 95% CI -0.2 to 1.2, p = 0.13). Analgesic consumptions were also similar (difference -18 mg diclofenac equivalents, 95% CI -43 mg to 7 mg, p = 0.17), with small initial differences diminishing over time. There were no differences between groups in any of the secondary outcomes and stratified analyses provided no evidence for potential benefits of SMT in specific patient groups. The extended follow-up showed similar patterns. CONCLUSIONS: SMT is unlikely to result in relevant early pain reduction in patients with acute low back pain.

Sensorimotor integration compensates for visual localization errors during smooth pursuit eye movements.

To localize a seen object, the CNS has to integrate the object's retinal location with the direction of gaze. Here we investigate this process by examining the localization of static objects during smooth pursuit eye movements. The normally experienced stability of the visual world during smooth pursuit suggests that the CNS essentially compensates for the eye movement when judging target locations. However, certain systematic localization errors are made, and we use these to study the process of sensorimotor integration. During an eye movement, a static object's image moves across the retina. Objects that produce retinal slip are known to be mislocalized: objects moving toward the fovea are seen too far on in their trajectory, whereas errors are much smaller for objects moving away from the fovea. These effects are usually studied by localizing the moving object relative to a briefly flashed one during fixation: moving objects are then mislocalized, but flashes are not. In our first experiment, we found that a similar differential mislocalization occurs for static objects relative to flashes during pursuit. This effect is not specific for horizontal pursuit but was also found in other directions. In a second experiment, we examined how this effect generalizes to positions outside the line of eye movement. We found that large localization errors were found in the entire hemifield ahead of the pursuit target and were predominantly aligned with the direction of eye movement. In a third experiment, we determined whether it is the flash or the static object that is mislocalized ahead of the pursuit target. In contrast to fixation conditions, we found that during pursuit it is the flash, not the static object, which is mislocalized. In a fourth experiment, we used egocentric localization to confirm this result. Our results suggest that the CNS compensates for the retinal localization errors to maintain position constancy for static objects during pursuit. This compensation is achieved in the process of sensorimotor integration of retinal and gaze signals: different retinal areas are integrated with different gaze signals to guarantee the stability of the visual world.

Bivalent disulfide-stabilized fragment variable immunotoxin directed against mesotheliomas and ovarian cancer.

We have used protein engineering to generate a stable bivalent fragment variable (Fv) molecule from the antimesothelin antibody SS, in which the VH and VL domains of the Fv are linked to each other by a disulfide bond, and the two Fvs are connected by a flexible 15-amino acid (Gly4-Ser)3 linker. The SS (dsFv)2 molecule is fused to a M(r) 38,000 truncated form of Pseudomonas exotoxin to generate a bivalent, disulfide stabilized, (dsFv)2 immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro, and purified to approximately 95% purity with a high yield of > 10%. Binding studies demonstrated that the (dsFv)2 molecule has 40 times higher apparent affinity for recombinant mesothelin than a monovalent dsFv molecule. The (dsFv)2 immunotoxin was 4-10-fold more cytotoxic to three mesothelin antigen-positive cell lines than the monovalent dsFv immunotoxin. However, when tested in mice bearing tumor cells expressing mesothelin, the antitumor activity of the bivalent immunotoxin is very similar to the activity of the lower affinity monovalent immunotoxin. Our data indicate that increasing affinity of an antibody fragment does not necessarily lead to higher antitumor activity of an immunotoxin in vivo.

A comparison of the cost-effectiveness of remifentanil versus fentanyl as an adjuvant to general anesthesia for outpatient gynecologic surgery.

The unique pharmacokinetic properties of remifentanil make it a potentially useful adjuvant during general anesthesia for ambulatory surgery. Fentanyl, inexpensive and easy to administer, is the most common opioid used for this purpose. As an adjuvant to general anesthesia for outpatient gynecologic surgery, we questioned if remifentanil was cost-effective as an alternative to fentanyl. Thirty-four patients undergoing gynecologic laparoscopy or hysteroscopy were prospectively and randomly assigned to a standard practice (n = 18) or a study (n = 16) group. Standard practice patients received fentanyl (3 microg/kg) before induction; study patients received remifentanil by continuous infusion (0.5 microg x kg. min(-1) at induction, then 0.2 microg x kg x min(-1)). Sevoflurane was titrated to a Bispectral index value of 40-55. We investigated recovery profiles, patient and health care professional satisfaction, and drug costs. The incidence of rescue antiemetic treatment (2 of 16 vs. 8 of 18; P = 0.013) and the nausea visual analog scale scores during second stage recovery (0.2 vs. 0.6; P = 0.044) were more frequent in the study group. However, the incidence of intraoperative adverse events and other postoperative sequelae, recovery times, pain and nausea visual analog scale scores, opioid analgesic dosage requirements in the postanesthetic care unit, and satisfaction survey responses were similar between groups. Perioperative drug costs per patient were $17.72 more in the remifentanil (vs. fentanyl) group.

Increased binding affinity enhances targeting of glioma xenografts by EGFRvIII-specific scFv.

Combinatorial variation of CDR3 of V(H) and V(L), followed by phage display, was used to select affinity mutants of the parental anti-epidermal growth factor receptor-vIII (EGFRvIII) scFv MR1. One mutant, MR1-1(scFv), had increased specific binding affinity for EGFRvIII. It was produced and radiolabeled, and its biodistribution was evaluated in human glioma-bearing athymic mice. MR1-1 targeted the same EGFRvIII epitope as MR1 with an approximately 15-fold higher affinity (K(d) = 1.5 x 10(-9) M) measured by surface resonance analysis. Labeling with (131)I or (125)I was performed, and the immunoreactive fraction of the labeled MR1-1(scFv) was 50% to 55%. After incubation at 37 degrees C for 4 days, the binding affinity was maintained at 60% of initial levels. The specificity of MR1-1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC-labeled scFv with the EGFRvIII-expressing U87MG. DeltaEGFR cell line or with the EGFRvIII-negative U87MG cell line in the presence or absence of competing unlabeled MR1-1(scFv). We also investigated the internalization and processing of MR1-1 compared with MR1; MR1-1 exhibited levels of both cell surface retention and internalization up to 5 times higher than those by MR1. In biodistribution studies performed in athymic mice bearing s.c. U87MG. DeltaEGFR tumor xenografts, animals received paired-label intratumoral infusions of (131)I-labeled MR1-1(scFv) and (125)I-labeled MR1(scFv). Our results showed an up to 244% +/- 77% increase in tumor uptake for MR1-1 compared with that for MR1. The improved tumor retention of MR1-1(scFv) combined with its rapid clearance from normal tissues also resulted in sustained higher tumor:normal organ ratios. These results suggest that the improved affinity of MR1-1 can significantly impact in vivo glioma-specific targeting and immunotherapy.

Site-specific expression of transferrin receptor by human colon cancer cells directly correlates with eradication by antitransferrin recombinant immunotoxin.

We determined the efficacy of HB21(Fv)PE40, a single-chain immunotoxin made by fusing the variable regions of a monoclonal antibody directed at the human transferrin receptor (TfR) with a truncated mutant of Pseudomonas exotoxin (PE), against metastatic human colon carcinoma KM12L4 cells growing in the liver or subcutis of nude mice. Organ-specific modulation of TfR expression was examined by immunohistochemistry and flow cytometry using anti-human CD71 antibody. KM12L4 cells expressed human TfR and were lysed in vitro by HB21(Fv)PE40 but not LMB-7 (a control immunotoxin specific for a Lewis Y-related carbohydrate antigen). KM12L4 cells growing in the liver expressed higher levels of TfR than cells growing s.c. Systemic administration of HB21(Fv)PE40 eliminated KM12L4 liver metastasis, whereas administration of LMB-7 did not. Treatment of mice with HB21(Fv)PE40 only delayed the growth of s.c. tumors. KM12L4 cells recovered from liver metastases, expressed higher levels of TfR, and were more sensitive to lysis by HB21(Fv)PE40 than KM12L4 cells recovered from s.c. tumors. Indeed, collectively, the data show that the expression level of the TfR by human colon cancer cells is modulated by the organ microenvironment which can be advantageous for the use of therapeutic immunotoxins.

Immunotoxins with increased activity against epidermal growth factor receptor vIII-expressing cells produced by antibody phage display.

Recombinant immunotoxins are fusion proteins composed of Fv regions of antibodies and bacterial or plant toxins that are being developed for the targeted therapy of cancer. MR1(Fv)-PE38 is a single-chain recombinant immunotoxin that targets a mutant form of the epidermal growth factor receptor (EGFR), EGFRvIII, that is frequently overexpressed in malignant glioblastomas. We have used random complementarity determining region (CDR) mutagenesis to obtain mutants of MR1(Fv) with an increased affinity for EGFRvIII and an increased activity when converted to a recombinant immunotoxin. Initially, nine residues of heavy chain CDR3 were randomly mutagenized, and several mutants with increased binding affinity were isolated. All mutations were located at amino acids 98 and 99, which correspond to a DNA hot spot, a DNA sequence that mutates at high frequency during natural antibody maturation. A specific region of variable region of antibody light chain CDR3 was mutagenized that corresponded to a hot spot and a mutant (MR1-1) with an additional increase in affinity, and cytotoxic activity was isolated. These studies show that targeting hot spots in the CDRs of Fvs is an effective approach to obtaining Fvs with increased affinity. The increased affinity of MR1-1(Fv) makes it an attractive candidate for the targeted therapy of glioblastomas.

Localization of a seen finger is based exclusively on proprioception and on vision of the finger.

In a previous study we investigated how the CNS combines simultaneous visual and proprioceptive information about the position of the finger. We found that localization of the index finger of a seen hand was more precise (a smaller variance) than could reasonably be expected from the precision of localization on the basis of vision only and proprioception only. This suggests that, in localizing the tip of the index finger of a seen hand, the CNS may make use of more information than proprioceptive information and visual information about the fingertip. In the present study we investigate whether this additional information stems from additional sources of sensory information. In experiment 1 we tested whether seeing an entire arm instead of only the fingertip gives rise to a more precise proprioceptive and/or visual localization of that fingertip. In experiment 2 we checked whether the presence of a structured visual environment leads to a more precise proprioceptive localization of the index finger of an unseen hand. In experiment 3 we investigated whether looking in the direction of the index finger of an unseen hand improves proprioceptive localization of that finger. We found no significant effect in any of the experiments. The results refute the hypothesis that the investigated effects can explain the previously reported very precise localization of a seen hand. This suggests that localization of a seen finger is based exclusively on proprioception and on vision of the finger. The results suggest that these sensory signals may contain more information than is described by the magnitude of their variances.

Integration of proprioceptive and visual position-information: An experimentally supported model.

To localize one's hand, i.e., to find out its position with respect to the body, humans may use proprioceptive information or visual information or both. It is still not known how the CNS combines simultaneous proprioceptive and visual information. In this study, we investigate in what position in a horizontal plane a hand is localized on the basis of simultaneous proprioceptive and visual information and compare this to the positions in which it is localized on the basis of proprioception only and vision only. Seated at a table, subjects matched target positions on the table top with their unseen left hand under the table. The experiment consisted of three series. In each of these series, the target positions were presented in three conditions: by vision only, by proprioception only, or by both vision and proprioception. In one of the three series, the visual information was veridical. In the other two, it was modified by prisms that displaced the visual field to the left and to the right, respectively. The results show that the mean of the positions indicated in the condition with both vision and proprioception generally lies off the straight line through the means of the other two conditions. In most cases the mean lies on the side predicted by a model describing the integration of multisensory information. According to this model, the visual information and the proprioceptive information are weighted with direction-dependent weights, the weights being related to the direction-dependent precision of the information in such a way that the available information is used very efficiently. Because the proposed model also can explain the unexpectedly small sizes of the variable errors in the localization of a seen hand that were reported earlier, there is strong evidence to support this model. The results imply that the CNS has knowledge about the direction-dependent precision of the proprioceptive and visual information.

The precision of proprioceptive position sense.

The purpose of this study was to determine the precision of proprioceptive localization of the hand in humans. We derived spatial probability distributions which describe the precision of localization on the basis of three different sources of information: proprioceptive information about the left hand, proprioceptive information about the right hand, and visual information. In the experiment subjects were seated at a table and had to perform three different position-matching tasks. In each task, the position of a target and the position of an indicator were available in a different combination of two of these three sources of information. From the spatial distributions of indicated positions in these three conditions, we derived spatial probability distributions for proprioceptive localization of the two hands and for visual localization. For proprioception we found that localization in the radial direction with respect to the shoulder is more precise than localization in the azimuthal direction. The distributions for proprioceptive localization also suggest that hand positions closer to the shoulder are localized more precisely than positions further away. These patterns can be understood from the geometry of the arm. In addition, the variability in the indicated positions suggests that the shoulder and elbow angles are known to the central nervous system with a precision of 0.6-1.1 degrees. This is a considerably better precision than the values reported in studies on perception of these angles. This implies that joint angles, or quantities equivalent to them, are represented in the central nervous system more precisely than they are consciously perceived. For visual localization we found that localization in the azimuthal direction with respect to the cyclopean eye is more precise than localization in the radial direction. The precision of the perception of visual direction is of the order of 0.2-0.6 degrees.

Improved stability and yield of a Fv-toxin fusion protein by computer design and protein engineering of the Fv.

The conversion of the anti-mesothelin monoclonal antibody K1 to a single-chain Fv (scFv) that is fused to a truncated form of Pseudomonas exotoxin A (PE) results in a fusion protein (immunotoxin) that is unstable and refolds very inefficiently. We have devised a method that identifies candidate residues in the framework region of K1 Fv that, when mutated, improved the yield and stability of the protein. The method works by initially aligning the framework sequences of K1 VH and VL with those of other scFvs that are stable and give a good yield as immunotoxins. Then we assigned a character to each residue that indicates its state of exposure based on the known crystal structures of Fabs. This identifies residues that are not compatible with their environment in the folded state of the protein. Next we calculated the frequencies of different amino acids for each position of the Fvs based on the available sequence database. This identifies residues that are not commonly present in the conserved positions. If these residues are compatible with their exposure profile they are left unaltered. Otherwise, they are identified as candidate residues for mutation. We identified two such residues in the VH (T82 and A85) and two in the VL (H36 and V60) of K1 that did not seem appropriate for their respective positions. By mutating these residues in K1 into those that occur most commonly in the sequence database or in stable scFvs, we significantly improved the stability and yield of the K1 scFv immunotoxins. By making single and combined mutations we assessed the relative contribution of mutations at these four sites towards the stability and yield of K1 scFv immunotoxins. The method we devised is probably general and can be used to improve other scFvs.

Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity.

Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers. Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents. In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin. When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice. After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells. The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A. The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37 degrees C and is very cytotoxic to cells expressing mesothelin. It also produces regressions of tumors expressing mesothelin. This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent. This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.

Cloning and expression of the recombinant FAb fragment of monoclonal antibody K1 that reacts with mesothelin present on mesotheliomas and ovarian cancers.

The monoclonal antibody (MAb) K1 recognizes an approximate 40 kDa glycoprotein, mesothelin, that is present on the surface of human mesothelial cells, mesotheliomas and ovarian cancers. We have cloned the cDNAs encoding the variable regions of MAb K1 and constructed plasmids for expression of recombinant K1(FAb). Recombinant FAb was produced in Escherichia coli in inclusion bodies that were solubilized and refolded to active protein. Binding of K1 MAb and FAb was compared by radioactive binding and competition assays and by surface plasmon resonance (BIAcore). Recombinant K1(FAb) binds to cells expressing K1-antigen with a similar affinity as papain derived FAb from K1(IgG) and with a 4- to 10-fold reduced affinity compared with bivalent IgG. The cloned FAb can be used to make higher affinity antibodies and immunoconjugates that could be useful for various types of immunotherapies.

Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation.

We have developed a novel method to stabilize a recombinant antibody Fv fragment. The V(H) and V(L) domains of this Fv fragment, called pFv (permutated Fv), are covalently interconnected to each other at the two "base-loops" that normally connect V(H) beta strand 3 to 3b and V(L) beta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, we connected the N-terminal half of the V(L) domain (V(L) 1-40) of murine antibody anti-Tac to the C-terminal half of V(H) (V(H) 42-115). We also fused the C terminus of V(H) by a (Gly4Ser)3 linker to the N-terminal half of V(H) (V(H) 1-40, thereby generating a permutated V(H) domain). Finally we connected the base loop of V(H) (N-terminal half) to the C-terminal half of V(L) (V(H) 42-115). The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxin. Fvs with the correct structure were produced by refolding of recombinant inclusion body protein using a renaturation protocol that was originally developed for Fab and scFv fragments. Due to the artificially connected and permutated primary sequence, the folding pathway for the pFv structure may possibly be different from the conventional folding of antibody domains. Analysis of antigen binding of anti-Tac pFv, and of the specific cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its affinity and full specificity when compared to anti-Tac scFv. Also anti-Tac pFv was relatively stable, retaining 25% of its binding activity after a 24 hour incubation in human serum at 37 degrees C. This indicates that connection of base loops can be a useful alternative to linker or disulfide stabilization of Fv fragments.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display.

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37 degrees C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.