Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

Genetic defects of gamma-secretase genes in a multiple myeloma patient with high and dysregulated BCMA surface density: A case report.

Multiple myeloma (MM) cells from 1 out of 20 patient expressed high basal levels of membrane B-cell maturation antigen (BCMA, TNFRSF17, CD269), which was not upregulated by gamma-secretase inhibitor, suggesting a defective BCMA shedding by gamma-secretase. Genetic analyses of the patient's bone marrow DNA showed no mutations within the BCMA coding region, but rather partial deletion of PSEN1 and amplification of PSEN2, which encode alternative catalytic units of gamma-secretase. Altogether the data suggest that pt#12 MM cells express high and dysregulated BCMA with no shedding, due to genetic alterations of one or more gamma-secretase subunits.

The use of new approach methodologies for the environmental risk assessment of food and feed chemicals.

New Approach Methodologies (NAMs) provide tools for supporting both human and environmental risk assessment (HRA and ERA). This short review provides recent insights regarding the use of NAMs in ERA of food and feed chemicals. We highlight the usefulness of tiered methods supporting weight-of-evidence approaches in relation to problem formulation (i.e., data availability, time, and resource availability). In silico models, including quantitative structure activity relationship models, support filling data gaps when no chemical property or ecotoxicological data are available, and biologically-based models (e.g., toxicokinetic-toxicodynamic models, dynamic energy models, physiologically-based models and species sensitivity distributions) are applicable in more data rich situations, including landscape-based modelling approaches. Particular attention is given to provide practical examples to apply the approaches described in real-world settings. We conclude with future perspectives, with regards to the need for addressing complex challenges such as chemical mixtures and multiple stressors in a wide range of organisms and ecosystems.

Risk Assessment of Combined Exposure to Multiple Chemicals at the European Food Safety Authority: Principles, Guidance Documents, Applications and Future Challenges.

Human health and animal health risk assessment of combined exposure to multiple chemicals use the same steps as single-substance risk assessment, namely problem formulation, exposure assessment, hazard assessment and risk characterisation. The main unique feature of combined RA is the assessment of combined exposure, toxicity and risk. Recently, the Scientific Committee of the European Food Safety Authority (EFSA) published two relevant guidance documents. The first one "Harmonised methodologies for the human health, animal health and ecological risk assessment of combined exposure to multiple chemicals" provides principles and explores methodologies for all steps of risk assessment together with a reporting table. This guidance supports also the default assumption that dose addition is applied for combined toxicity of the chemicals unless evidence for response addition or interactions (antagonism or synergism) is available. The second guidance document provides an account of the scientific criteria to group chemicals in assessment groups using hazard-driven criteria and prioritisation methods, i.e., exposure-driven and risk-based approaches. This manuscript describes such principles, provides a brief description of EFSA's guidance documents, examples of applications in the human health and animal health area and concludes with a discussion on future challenges in this field.

A Tiered Approach for Assessing Individual and Combined Risk of Pyrethroids Using Human Biomonitoring Data.

Pyrethroids are a major insecticide class, suitable for biomonitoring in humans. Due to similarities in structure and metabolic pathways, urinary metabolites are common to various active substances. A tiered approach is proposed for risk assessment. Tier I was a conservative screening for overall pyrethroid exposure, based on phenoxybenzoic acid metabolites. Subsequently, probabilistic approaches and more specific metabolites were used for refining the risk estimates. Exposure was based on 95th percentiles from HBM4EU aligned studies (2014-2021) covering children in Belgium, Cyprus, France, Israel, Slovenia, and The Netherlands and adults in France, Germany, Israel, and Switzerland. In all children populations, the 95th percentiles for 3-phenoxybenzoic acid (3-PBA) exceeded the screening value. The probabilistic refinement quantified the risk level of the most exposed population (Belgium) at 2% or between 1-0.1% depending on the assumptions. In the substance specific assessments, the 95th percentiles of urinary concentrations in the aligned studies were well below the respective human biomonitoring guidance values (HBM-GVs). Both information sets were combined for refining the combined risk. Overall, the HBM data suggest a low health concern, at population level, related to pyrethroid exposure for the populations covered by the studies, even though a potential risk for highly exposed children cannot be completely excluded. The proposed tiered approach, including a screening step and several refinement options, seems to be a promising tool of scientific and regulatory value in future.

Role of Fc Core Fucosylation in the Effector Function of IgG1 Antibodies.

The presence of fucose on IgG1 Asn-297 N-linked glycan is the modification of the human IgG1 Fc structure with the most significant impact on FcÉ£RIII affinity. It also significantly enhances the efficacy of antibody dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in vitro, induced by IgG1 therapeutic monoclonal antibodies (mAbs). The effect of afucosylation on ADCC or antibody dependent phagocytosis (ADCP) mediated by macrophages or polymorphonuclear neutrophils (PMN) is less clear. Evidence for enhanced efficacy of afucosylated therapeutic mAbs in vivo has also been reported. This has led to the development of several therapeutic antibodies with low Fc core fucose to treat cancer and inflammatory diseases, seven of which have already been approved for clinical use. More recently, the regulation of IgG Fc core fucosylation has been shown to take place naturally during the B-cell immune response: A decrease in α-1,6 fucose has been observed in polyclonal, antigen-specific IgG1 antibodies which are generated during alloimmunization of pregnant women by fetal erythrocyte or platelet antigens and following infection by some enveloped viruses and parasites. Low IgG1 Fc core fucose on antigen-specific polyclonal IgG1 has been linked to disease severity in several cases, such as SARS-CoV 2 and Dengue virus infection and during alloimmunization, highlighting the in vivo significance of this phenomenon. This review aims to summarize the current knowledge about human IgG1 Fc core fucosylation and its regulation and function in vivo, in the context of both therapeutic antibodies and the natural immune response. The parallels in these two areas are informative about the mechanisms and in vivo effects of Fc core fucosylation, and may allow to further exploit the desired properties of this modification in different clinical contexts.

Improving the Risk Assessment of Pesticides through the Integration of Human Biomonitoring and Food Monitoring Data: A Case Study for Chlorpyrifos.

The risk assessment of pesticide residues in food is a key priority in the area of food safety. Most jurisdictions have implemented pre-marketing authorization processes, which are supported by prospective risk assessments. These prospective assessments estimate the expected residue levels in food combining results from residue trials, resembling the pesticide use patterns, with food consumption patterns, according to internationally agreed procedures. In addition, jurisdictions such as the European Union (EU) have implemented large monitoring programs, measuring actual pesticide residue levels in food, and are supporting large-scale human biomonitoring programs for confirming the actual exposure levels and potential risk for consumers. The organophosphate insecticide chlorpyrifos offers an interesting case study, as in the last decade, its acceptable daily intake (ADI) has been reduced several times following risk assessments by the European Food Safety Authority (EFSA). This process has been linked to significant reductions in the use authorized in the EU, reducing consumers' exposure progressively, until the final ban in 2020, accompanied by setting all EU maximum residue levels (MRL) in food at the default value of 0.01 mg/kg. We present a comparison of estimates of the consumer's internal exposure to chlorpyrifos based on the urinary marker 3,5,6-trichloro-2-pyridinol (TCPy), using two sources of monitoring data: monitoring of the food chain from the EU program and biomonitoring of European citizens from the HB4EU project, supported by a literature search. Both methods confirmed a drastic reduction in exposure levels from 2016 onwards. The margin of exposure approach is then used for conducting retrospective risk assessments at different time points, considering the evolution of our understanding of chlorpyrifos toxicity, as well as of exposure levels in EU consumers following the regulatory decisions. Concerns are presented using a color code, and have been identified for almost all studies, particularly for the highest exposed group, but at different levels, reaching the maximum level, red code, for children in Cyprus and Israel. The assessment uncertainties are highlighted and integrated in the identification of levels of concern.

Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use.

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

Identification of Human SARS-CoV-2 Monoclonal Antibodies from Convalescent Patients Using EBV Immortalization.

We report the isolation of two human IgG1k monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike protein. These mAbs were isolated from two donors who had recovered from COVID-19 infection during the first pandemic peak in the Lombardy region of Italy, the first European and initially most affected region in March 2020. We used the method of EBV immortalization of purified memory B cells and supernatant screening with a spike S1/2 assay for mAb isolation. This method allowed rapid isolation of clones, with one donor showing about 7% of clones positive against spike protein, whereas the other donor did not produce positive clones out of 91 tested. RNA was extracted from positive clones 39-47 days post-EBV infection, allowing VH and VL sequencing. The same clones were sequenced again after a further 100 days in culture, showing that no mutation had taken place during in vitro expansion. The B cell clones could be expanded in culture for more than 4 months after EBV immortalization and secreted the antibodies stably during that time, allowing to purify mg quantities of each mAb for functional assays without generating recombinant proteins. Unfortunately, neither mAb had significant neutralizing activity in a virus infection assay with several different SARS-CoV-2 isolates. The antibody sequences are made freely available.

Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B cells opsonized with anti-CD20 antibodies.

Polymorphonuclear neutrophils (PMNs) have previously been reported to mediate phagocytosis of anti-CD20-opsonized B cells from patients with chronic lymphocytic leukemia (CLL). However, recent data have suggested that PMNs, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and to discriminate between trogocytosis and phagocytosis. In live-cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMNs of anti-CD20-opsonized CLL B cells, but could detect only the repeated close contact between effectors and targets, which suggested trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we observed that a mean of 50% and 75% of PMNs had taken a fraction of the dye from CLL B cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis occurs, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B cells, which was evident with rituximab but not obinutuzumab. We conclude that PMNs mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B cells, and we discuss the implications of this finding in patients with CLL treated with rituximab or obinutuzumab in vivo.

Two-photon polymerized "nichoid" substrates maintain function of pluripotent stem cells when expanded under feeder-free conditions.

BACKGROUND: The use of pluripotent cells in stem cell therapy has major limitations, mainly related to the high costs and risks of exogenous conditioning and the use of feeder layers during cell expansion passages. METHODS: We developed an innovative three-dimensional culture substrate made of "nichoid" microstructures, nanoengineered via two-photon laser polymerization. The nichoids limit the dimension of the adhering embryoid bodies during expansion, by counteracting cell migration between adjacent units of the substrate by its microarchitecture. We expanded mouse embryonic stem cells on the nichoid for 2 weeks. We compared the expression of pluripotency and differentiation markers induced in cells with that induced by flat substrates and by a culture layer made of kidney-derived extracellular matrix. RESULTS: The nichoid was found to be the only substrate, among those tested, that maintained the expression of the OCT4 pluripotency marker switched on and, simultaneously, the expression of the differentiation markers GATA4 and α-SMA switched off. The nichoid promotes pluripotency maintenance of embryonic stem cells during expansion, in the absence of a feeder layer and exogenous conditioning factors, such as the leukocyte inhibitory factor. CONCLUSIONS: We hypothesized that the nichoid microstructures induce a genetic reprogramming of cells by controlling their cytoskeletal tension. Further studies are necessary to understand the exact mechanism by which the physical constraint provided by the nichoid architecture is responsible for cell reprogramming. The nichoid may help elucidate mechanisms of pluripotency maintenance, while potentially cutting the costs and risks of both feed-conditioning and exogenous conditioning for industrial-scale expansion of stem cells.

Design of a cone-and-plate device for controlled realistic shear stress stimulation on endothelial cell monolayers.

Endothelial cells are constantly exposed to blood flow and the resulting frictional force, the wall shear stress, varies in magnitude and direction with time, depending on vasculature geometry. Previous studies have shown that the structure and function of endothelial cells, and ultimately of the vessel wall, are deeply affected by the nature of wall shear stress waveforms. To investigate the in vitro effects of these stimuli, we developed a compact, programmable, real-time operated system based on cone-and-plate geometry, that can be used within a standard cell incubator. To verify the capability to replicate realistic shear stress waveforms, we calculated both analytically and numerically to what extent the system is able to correctly deliver the stimuli defined by the user at plate level. Our results indicate that for radii greater than 25 mm, the shear stress is almost uniform and directly proportional to cone rotation velocity. We further established that using a threshold of 10 Hz of wall shear stress waveform frequency components, oscillating flow conditions can be reproduced on cell monolayer surface. Finally, we verified the capability of the system to perform long-term flow exposure experiments ensuring sterility and cell culture viability on human umbilical vein endothelial cells exposed to unidirectional and oscillating shear stress. In conclusion, the system we developed is a highly dynamic, easy to handle, and able to generate pulsatile and unsteady oscillating wall shear stress waveforms. This system can be used to investigate the effects of realistic stimulations on endothelial cells, similar to those exerted in vivo by blood flow.

Endothelial cell activation by hemodynamic shear stress derived from arteriovenous fistula for hemodialysis access.

Intimal hyperplasia (IH) is the first cause of failure of an arteriovenous fistula (AVF). The aim of the present study was to investigate the effects on endothelial cells (ECs) of shear stress waveforms derived from AVF areas prone to develop IH. We used a cone-and-plate device to obtain real-time control of shear stress acting on EC cultures. We exposed human umbilical vein ECs for 48 h to different shear stimulations calculated in a side-to-end AVF model. Pulsatile unidirectional flow, representative of low-risk stenosis areas, induced alignment of ECs and actin fiber orientation with flow. Shear stress patterns of reciprocating flow, derived from high-risk stenosis areas, did not affect EC shape or cytoskeleton organization, which remained similar to static cultures. We also evaluated flow-induced EC expression of genes known to be involved in cytoskeletal remodeling and expression of cell adhesion molecules. Unidirectional flow induced a significant increase in Kruppel-like factor 2 mRNA expression, whereas it significantly reduced phospholipase D1, α4-integrin, and Ras p21 protein activator 1 mRNA expression. Reciprocating flow did not increase Kruppel-like factor 2 mRNA expression compared with static controls but significantly increased mRNA expression of phospholipase D1, α4-integrin, and Ras p21 protein activator 1. Reciprocating flow selectively increased monocyte chemoattractant protein-1 and IL-8 production. Furthermore, culture medium conditioned by ECs exposed to reciprocating flows selectively increased smooth muscle cell proliferation compared with unidirectional flow. Our results indicate that protective vascular effects induced in ECs by unidirectional pulsatile flow are not induced by reciprocating shear forces, suggesting a mechanism by which oscillating flow conditions may induce the development of IH in AVF and vascular access dysfunction.

Regression of Renal Disease by Angiotensin II Antagonism Is Caused by Regeneration of Kidney Vasculature.

Chronic renal insufficiency inexorably progresses in patients, such as it does after partial renal ablation in rats. However, the progression of renal diseases can be delayed by angiotensin II blockers that stabilize renal function or increase GFR, even in advanced phases of the disease. Regression of glomerulosclerosis can be induced by angiotensin II antagonism, but the effect of these treatments on the entire vascular tree is unclear. Here, using microcomputed tomography and scanning electron microscopy, we compared the size and extension of kidney blood vessels in untreated Wistar rats with those in untreated and angiotensin II antagonist-treated Munich Wistar Frömter (MWF) rats that spontaneously develop kidney disease with age. The kidney vasculature underwent progressive rarefaction in untreated MWF rats, substantially affecting intermediate and small vessels. Microarray analysis showed increased Tgf-ß and endothelin-1 gene expression with age. Notably, 10-week inhibition of the renin-angiotensin system regenerated kidney vasculature and normalized Tgf-ß and endothelin-1 gene expression in aged MWF rats. These changes were associated with reduced apoptosis, increased endothelial cell proliferation, and restoration of Nrf2 expression, suggesting mechanisms by which angiotensin II antagonism mediates regeneration of capillary segments. These results have important implications in the clinical setting of chronic renal insufficiency.

Small diameter electrospun silk fibroin vascular grafts: Mechanical properties, in vitro biodegradability, and in vivo biocompatibility.

To overcome the drawbacks of autologous grafts currently used in clinical practice, vascular tissue engineering represents an alternative approach for the replacement of small diameter blood vessels. In the present work, the production and characterization of small diameter tubular matrices (inner diameter (ID)=4.5 and 1.5 mm), obtained by electrospinning (ES) of Bombyx mori silk fibroin (SF), have been considered. ES-SF tubular scaffolds with ID=1.5 mm are original, and can be used as vascular grafts in pediatrics or in hand microsurgery. Axial and circumferential tensile tests on ES-SF tubes showed appropriate properties for the specific application. The burst pressure and the compliance of ES-SF tubes were estimated using the Laplace's law. Specifically, the estimated burst pressure was higher than the physiological pressures and the estimated compliance was similar or higher than that of native rat aorta and Goretex® prosthesis. Enzymatic in vitro degradation tests demonstrated a decrease of order and crystallinity of the SF outer surface as a consequence of the enzyme activity. The in vitro cytocompatibility of the ES-SF tubes was confirmed by the adhesion and growth of primary porcine smooth muscle cells. The in vivo subcutaneous implant into the rat dorsal tissue indicated that ES-SF matrices caused a mild host reaction. Thus, the results of this investigation, in which comprehensive morphological and mechanical aspects, in vitro degradation and in vitro and in vivo biocompatibility were considered, indicate the potential suitability of these ES-SF tubular matrices as scaffolds for the regeneration of small diameter blood vessels.

In vivo regeneration of elastic lamina on fibroin biodegradable vascular scaffold.

PURPOSE: There is an increasing need for vascular grafts in the field of surgical revascularization. Artificial grafts offer alternative strategies to autologous tissue, however, small caliber (diameter <6 mm) vascular prosthesis are associated with a high incidence of thrombosis and early failure. Despite promising results, vascular tissue engineering is not yet a clinical reality due to the complexity of this approach. We aimed at investigating the use of fibroin, a biodegradable protein derived from silk, as an acellular vascular graft for in vivo recellularization. 
 METHODS: We produced small caliber fibroin matrices by electrospinning to replace small arterial segments. Electrospun fibroin scaffolds were implanted into the abdominal aorta of Lewis rats by end-to-end anastomosis. Seven days after implantation, fibroin matrices were recovered and processed for histological and immunohistochemical analysis.
 RESULTS: Fibroin matrices allowed host cell infiltration, extracellular matrix remodeling, and ensured good patency of the grafts in the short term. Endothelial cells and smooth muscle cells were present in the explanted construct. Development of an elastic lamina adjacent to the lumen of the scaffold was observed with organization of intima and media layers. Vasa-vasorum were also present in the outer layer of the fibroin material.
 CONCLUSIONS: Our results indicate that formation of vascular tissue containing elastin occurs already at 7 days after implantation on fibroin scaffold without in vitro cellularization. The use of an acellular electrospun silk fibroin tubular scaffold could be a promising strategy for in vivo regeneration of vascular tissue in the clinical reality.

Shear stress reverses dome formation in confluent renal tubular cells.

BACKGROUND/AIMS: It has been shown that MDCK cells, a cell line derived from canine renal tubules, develop cell domes due to fluid pumped under cell monolayer and focal detachment from the adhesion surface. In vitro studies have shown that primary cilia of kidney tubular epithelial cells act as mechanosensors, increasing intracellular calcium within seconds upon changes in fluid shear stress (SS) on cell membrane. We then studied the effect of prolonged SS exposure on cell dome formation in confluent MDCK cell monolayers. METHODS: A parallel plate flow chamber was used to apply laminar SS at 2 dynes/cm(2) to confluent cell monolayers for 6 hours. Control MDCK cell monolayers were maintained in static condition. The effects of Ca(2+) blockade and cell deciliation on SS exposure were also investigated. RESULTS: Seven days after reaching confluence, static cultures developed liquid filled domes, elevating from culture plate. Exposure to SS induced almost complete disappearance of cell domes (0.4±0.8 vs. 11.4±2.8 domes/mm(2), p < 0.01, n=14). SS induced dome disappearance took place within minutes to hours, as shown by time-lapse videomicroscopy. Exposure to SS importantly affected cell cytoskeleton altering actin stress fibers expression and organization, and the distribution of tight junction protein ZO-1. Dome disappearance induced by flow was completely prevented in the presence of EGTA or after cell deciliation. CONCLUSIONS: These data indicate that kidney tubular cells are sensitive to apical flow and that these effects are mediated by primary cilia by regulation of Ca(2+) entry in to the cell. SS induced Ca(2+) entry provokes contraction of cortical actin ring that tenses cell-cell borders and decreases basal stress fibers. These processes may increase paracellular permeability and decrease basal adhesion making dome disappear. Elucidation of the effects of apical fluid flow on tubular cell function may open new insights on the pathophysiology of kidney diseases associated with cilia dysfunction.

Effect of inborn pancreatic islet deficit in the Munich Wister Frömter rat.

The total mass of pancreatic islet cells is a critical factor in glucose metabolic control. The aim of the present study was to investigate whether in the Munich Wistar Frömter (MWF) rat, beside a reduction in the number of nephrons, there are also alterations in the number of pancreatic islets and of ß cell mass. We also examined glucose metabolism, both in normal conditions and following intravenous glucose injection. The number of islets per pancreas, estimated by morphometrical analysis, was significantly lower in MWF rats than in Wistar rats (3,501±1,285 vs. 7,259±2,330 islet/rat, respectively). Also the mean number of islets per gram of body weight was significantly lower in MWF rats than in Wistar rats (18±7 in MWF rats vs. 28±10 islets/g bw in Wistar rats). Morphometric analysis of ß cell mass showed an average of 77.1±7% islet cells staining for insulin in MWF rats and 83.9±2.1% in the control Wistar rats. Despite the lower number of islets and ß cells, MWF and Wistar rats had comparable fasting blood glucose levels but significant differences in blood glucose following an intraperitoneal glucose tolerance test. In summary, pancreatic islets of MWF and Wistar rats showed a marked difference in morphometrical characteristics. While this difference is not associated with blood glucose levels, glucose metabolism after IPGTT between MWF and Wistar rats is significantly different. These data suggest that an inborn deficit in ß cell mass of about 60% is responsible for altered glucose metabolism and could favor the development of diabetes.

Effect of islet transplantation on metabolic glucose control in rats with diabetes.

BACKGROUND: Transplantation of pancreatic islets has been extensively investigated as a strategy for glycemic control in experimental animals and in patients with diabetes. We investigated whether islet transplantation allows us to obtain adequate islet function during glucose stimulation using a continuous glucose monitoring system (CGMS) in the rat. METHODS: We investigated four groups of eight rats each: healthy rats (controls), rats with diabetes, and rats with diabetes transplanted with microencapsulated islets in the peritoneal cavity or transplanted with free islets under the kidney capsule. Syngeneic islets were isolated from Lewis rats. After diabetes induction and islet implantation, when glycemia was stable, a glucose sensor was implanted, and an intraperitoneal glucose tolerance test (IPGTT) was performed to evaluate islet function. Interstitial glucose levels were analyzed, using a theoretical model, to estimate kinetics of glucose metabolism. RESULTS: Islet transplantation was effective in inducing normoglycemia in both groups, but results of IPGTTs showed that in animals with islets transplanted in microcapsules values of area under the curve and total glucose elimination constant (k(tot)) were significantly different from those in control animals and that these differences were even more important in animals with islets implanted under the kidney capsule. CONCLUSIONS: Our present investigation demonstrates that the application of CGMS was effective in evaluation of glucose metabolism by islet transplantation and indicates that efficient diabetes control can be achieved with this technology.