Randomized Bendamustine-Rituximab(R) + Bortezomib Induction and R + Lenalidomide Maintenance for Mantle Cell Lymphoma.

While initial therapy of mantle cell lymphoma (MCL) is not standardized, bendamustine-rituximab (BR) is commonly used in older patients. Rituximab (R) maintenance following induction is often utilized. Thus, the open-label, randomized phase II ECOG-ACRIN Cancer Research Group E1411 trial was designed to test two questions: 1) Does addition of bortezomib to BR induction (BVR) and/or 2) addition of lenalidomide to rituximab (LR) maintenance improve progression-free survival (PFS) in patients with treatment-naïve MCL? From 2012-2016, 373 previously untreated patients, 87% ≥ 60 years old, were enrolled in this trial. At a median follow up of 7.5 years, there is no difference in the median PFS of BR compared to BVR (5.5 yrs vs. 6.4 yrs, HR 0.90, 90% CI 0.70, 1.16). There were no unexpected additional toxicities with BVR treatment compared to BR, with no impact on total dose/duration of treatment received. Independent of the induction treatment, addition of lenalidomide to rituximab did not significantly improve PFS, with median PFS in R vs LR (5.9 yrs vs 7.2 yrs, HR 0.84 90% CI 0.62, 1.15). The majority of patients completed the planned 24 cycles of LR at the scheduled dose. In summary, adding bortezomib to BR induction does not prolong PFS in treatment-naïve MCL, and LR maintenance was not associated with longer PFS compared with rituximab alone following BR. Nonetheless, the > 5 year median PFS outcomes in this prospective cooperative group trial indicate the efficacy of BR followed by rituximab maintenance as highly effective initial therapy for older MCL patients. (NCT01415752).

Windborne long-distance migration of malaria mosquitoes in the Sahel.

Over the past two decades efforts to control malaria have halved the number of cases globally, yet burdens remain high in much of Africa and the elimination of malaria has not been achieved even in areas where extreme reductions have been sustained, such as South Africa1,2. Studies seeking to understand the paradoxical persistence of malaria in areas in which surface water is absent for 3-8 months of the year have suggested that some species of Anopheles mosquito use long-distance migration3. Here we confirm this hypothesis through aerial sampling of mosquitoes at 40-290 m above ground level and provide-to our knowledge-the first evidence of windborne migration of African malaria vectors, and consequently of the pathogens that they transmit. Ten species, including the primary malaria vector Anopheles coluzzii, were identified among 235 anopheline mosquitoes that were captured during 617 nocturnal aerial collections in the Sahel of Mali. Notably, females accounted for more than 80% of all of the mosquitoes that we collected. Of these, 90% had taken a blood meal before their migration, which implies that pathogens are probably transported over long distances by migrating females. The likelihood of capturing Anopheles species increased with altitude (the height of the sampling panel above ground level) and during the wet seasons, but variation between years and localities was minimal. Simulated trajectories of mosquito flights indicated that there would be mean nightly displacements of up to 300 km for 9-h flight durations. Annually, the estimated numbers of mosquitoes at altitude that cross a 100-km line perpendicular to the prevailing wind direction included 81,000 Anopheles gambiae sensu stricto, 6 million A. coluzzii and 44 million Anopheles squamosus. These results provide compelling evidence that millions of malaria vectors that have previously fed on blood frequently migrate over hundreds of kilometres, and thus almost certainly spread malaria over these distances. The successful elimination of malaria may therefore depend on whether the sources of migrant vectors can be identified and controlled.

Intravenous administration of multipotent stromal cells and bone allograft modification to enhance allograft healing.

Aim: This study investigated a coordinated strategy of revitalizing bone allograft with circulating multipotent stromal cells (MSCs). Materials & methods: After chemotactic and releasing assessments, stromal cell-derived factor 1 and platelet-derived growth factor BB in copolymers were coated on the bone allograft (AlloS-P). Allograft coated with copolymers alone (Allo), as controls, or AlloS-P was implanted into the femur of athymic mice, which received intravenous injections of human MSCs or saline at weeks 1, 2 and 3. Results: At week 8, the total callus volume (both cartilaginous and bony callus) around the allograft was the largest in the AlloS-P + MSC group (p < 0.05). Conclusion: Coating bone allograft with stromal cell-derived factor 1 and platelet-derived growth factor BB and intravenous injections of MSCs improved allograft incorporation.

Compromised Antibacterial Function of Multipotent Stromal Cells in Diabetes.

In diabetes, multipotent stromal cells (MSCs) are functionally deficient. It is unknown, however, whether their antibacterial function is compromised. In this study, MSCs were isolated from the bone marrow samples provided by nine diabetic and six nondiabetic donors and treated with or without Escherichia coli lipopolysaccharides (LPS). The supernatant of diabetic MSCs (MSCs-dia) and nondiabetic control MSCs (MSCs-c) was added into the cultures of E. coli for evaluation of the effect of MSCs-dia and MSCs-c on bacterial growth. The number of E. coli colonies increased when they were cultured with the supernatant of MSCs-dia, with or without LPS stimulation, compared with the E. coli cultured with the supernatant of MSCs-c. Human macrophages were co-cultured with either MSCs-dia or MSCs-c, for 24 h, and then cultured with heat-inactivated E. coli. Bacterial phagocytosis was reduced after macrophages were co-cultured with MSCs-dia. Gene expression of antibacterial peptide LL-37 and indoleamine 2,3-dioxygenase (IDO) by MSCs-dia was reduced compared with MSCs-c. The supernatant of MSCs-dia and MSCs-c was applied to a 42-cytokine antibody array. While the cytokine profiles of MSCs-dia and MSCs-c were largely similar, the productions of MCP-1 and interleukin-6 distinguished MSCs-dia from MSCs-c in response to LPS treatment. In conclusion, MSCs-dia were less inhibitive of the growth of bacteria and compromised in regulation of macrophages for bacterial phagocytosis. The reduced expression of IDO and LL-37 and an altered cytokine profile in MSCs-dia should be taken into consideration in developing cell therapies for diabetic infection.

Orthotopic Transplantation of Achilles Tendon Allograft in Rats: With or without Incorporation of Autologous Mesenchymal Stem Cells.

The biology and function of orthotopic transplantation of Achilles tendon allograft are unknown. Particularly, the revitalization of Achilles allograft is a clinical concern. Achilles allografts were harvested from donor rats and stored at -80 °C. Subcutaneous adipose tissue was harvested from the would-be allograft recipient rats for isolation of mesenchymal stem cells (MSCs). MSCs were cultured with growth differentiation factor-5 (GDF-5) and applied onto Achilles allografts on the day of transplantation. After the native Achilles tendon was resected from the left hind limb of the rats, Achilles allograft, with or without autologous MSCs, was implanted and sutured with calf muscles proximally and calcaneus distally. Animal gait was recorded presurgery and postsurgery weekly. The animals were sacrificed at week 4, and the transplanted Achilles allografts were collected for biomechanical testing and histology. The operated limbs had altered gait. By week 4, the paw print intensity, stance time, and duty cycle (percentage of the stance phase in a step cycle) of the reconstructed limbs were mostly recovered to the baselines recorded before surgery. Maximum load of failure was not different between Achilles allografts, with or without MSCs, and the native tendons. The Achilles allograft supplemented with MSCs had higher cellularity than the Achilles allograft without MSCs. Deposition of fine collagen (type III) fibers was active in Achilles allograft, with or without MSCs, but it was more evenly distributed in the allografts that were incubated with MSCs. In conclusion, orthotopically transplanted Achilles allograft healed with host tissues, regained strength, and largely restored Achilles function in 4 wk in rats. It is therefore a viable option for the reconstruction of a large Achilles tendon defect. Supplementation of MSCs improved repopulation of Achilles allograft, but large animal models, with long-term follow up and cell tracking, may be required to fully appreciate the functional benefits of MSCs.

Phase II trial of preoperative pemetrexed plus carboplatin in patients with stage IB-III nonsquamous non-small cell lung cancer (NSCLC).

OBJECTIVES: The combination of pemetrexed and carboplatin is a standard first-line treatment for patients with advanced NSCLC. In this pilot phase II trial, we evaluated the feasibility of using pemetrexed and carboplatin as neoadjuvant therapy, prior to definitive surgical resection, for patients with localized NSCLC. PATIENTS AND METHODS: Patients with potentially resectable, previously untreated, clinical stage IB-III, nonsquamous NSCLC were eligible for this trial. All patients received 4 cycles of pemetrexed (500 mg/m2) and carboplatin (AUC 6.0) administered at 21 day intervals. Three to 6 weeks after completion of chemotherapy, definitive surgical resection was attempted. The primary endpoint of this trial was the 3-year survival rate. RESULTS: Forty-six patients began protocol treatment, and 40 completed 4 courses of pemetrexed/carboplatin. Surgical resection was performed in 27 patients (59%); all had pathologic partial responses. The estimated 3-year survival rate for the entire group was 46%. Toxicity of neoadjuvant therapy was consistent with toxicity previously reported with pemetrexed/carboplatin. CONCLUSIONS: Administration of 4 courses of pemetrexed/carboplatin was feasible. The efficacy was similar to neoadjuvant regimens previously investigated. A significant number of patients 19 of 46 (41%) in this trial did not have surgical resection after neoadjuvant therapy. Further investigation of the role of neoadjuvant pemetrexed/carboplatin requires a larger, randomized clinical trial.

Functionally compromised synovium-derived mesenchymal stem cells in Charcot neuroarthropathy.

Charcot neuroarthropathy (CNA) often presents as a diabetic foot complication. The role of synovial mesenchymal stem cells (syn-MSCs) in the pathogenesis of CNA is unclear. Synovial samples were collected, for isolation of syn-MSCs, from diabetic patients with CNA (n=7) and non-diabetic patients with intra-articular fracture or normal joints (non-CNA; n=7) during foot surgery. The syn-MSCs in the CNA and non-CNA groups were characterized comparatively. The average number of colonies formed in the CNA group was 6±3.5 per half plate (10mm in diameter), while it was 43±21.6 in the non-CNA group (p<0.05). The average size (pixels) of the colonies in the CNA group was smaller than that in the non-CNA group. When the colonies were stratified into high-, medium- and low-density subgroups, colonies in the high-density subgroup of the CNA group were reduced in density. Expression of PPAR-γ, RUNX2, Sox9 and type II collagen by syn-MSCs in the CNA group was decreased during adipogenic, osteogenic and chondrogenic differentiation as compared with the non-CNA group. In conclusion, syn-MSCs in CNA joints were reduced in number, with declined differentiation potentials. The high-density subpopulation of the syn-MSCs was particularly affected by the pathology of CNA.

Effect of Low-Intensity Pulsed Ultrasound on Joint Injury and Post-Traumatic Osteoarthritis: an Animal Study.

This study investigated the therapeutic potential of low-intensity pulsed ultrasound (LIPUS) in post-traumatic osteoarthritis (PTOA). Intra-articular fracture of the medial tibial plateau was surgically created in 30 rats. LIPUS was applied to the operated joints either for the first 2 wk (LIPUS1-2 group) or in weeks 4 and 5 after intra-articular fracture (LIPUS4-5 group). In controls, the operated knees were not treated with LIPUS (LIPUS0 group). The rats were monitored with weekly gait analysis and euthanized at week 8. Among the altered gait parameters, the maximal and average paw print areas in the LIPUS1-2 and LIPUS4-5 groups, but not the LIPUS0 group, had either reached baseline or significantly recovered (70%, p <0.05) by week 8. PTOA pathology in both the LIPUS1-2 and LIPUS4-5 groups was less severe than that in the LIPUS0 group (Mankin score: 5.4 and 4.5 vs. 8.8, p <0.05). In conclusion, LIPUS treatment partially improved the gait of the affected limbs and reduced cartilage degeneration in PTOA.

A Surgical Model of Posttraumatic Osteoarthritis With Histological and Gait Validation.

BACKGROUND: Posttraumatic osteoarthritis (PTOA) is secondary to an array of joint injuries. Animal models are useful tools for addressing the uniqueness of PTOA progression in each type of joint injury and developing strategies for PTOA prevention and treatment. HYPOTHESIS: Intra-articular fracture induces PTOA pathology. STUDY DESIGN: Descriptive laboratory study. METHODS: Through a parapatellar incision, the medial tibial plateau was exposed in the left knees of 8 Sprague-Dawley rats. Osteotomy at the midpoint between the tibial crest and the outermost portion of the medial tibial plateau, including the covering articular cartilage, was performed using a surgical blade. The fractured medial tibial plateau was fixed with 2 needles transversely. The fractured knees were not immobilized. Before and after surgery, rat gait was recorded. Rats were sacrificed at week 8, and their knees were harvested for histology. RESULTS: After intra-articular fracture, the affected limbs altered gait from baseline (week 0). In the first 2 weeks, the gait of the operated limbs featured a reduced paw print intensity and stride length but increased maximal contact and stance time. Reduction of maximal and mean print area and duty cycle (the percentage of stance phase in a step) was present from week 1 to week 5. Only print length was reduced in weeks 7 and 8. At week 8, histology of the operated knees demonstrated osteoarthritic pathology. The severity of the PTOA pathology did not correlate with the changes of print length at week 8. CONCLUSION: Intra-articular fracture of the medial tibial plateau effectively induced PTOA in rat knees. During PTOA development, the injured limbs demonstrated characteristic gait. CLINICAL RELEVANCE: Intra-articular fracture represents severe joint injury and associates with a high rate of PTOA. This animal model, with histologic and gait validations, can be useful for future studies of PTOA prevention and early diagnosis.

Salinity-induced changes in gene expression from anterior and posterior gills of Callinectes sapidus (Crustacea: Portunidae) with implications for crustacean ecological genomics.

Decapods represent one of the most ecologically diverse taxonomic groups within crustaceans, making them ideal to study physiological processes like osmoregulation. However, prior studies have failed to consider the entire transcriptomic response of the gill - the primary organ responsible for ion transport - to changing salinity. Moreover, the molecular genetic differences between non-osmoregulatory and osmoregulatory gill types, as well as the hormonal basis of osmoregulation, remain underexplored. Here, we identified and characterized differentially expressed genes (DEGs) via RNA-Seq in anterior (non-osmoregulatory) and posterior (osmoregulatory) gills during high to low salinity transfer in the blue crab Callinectes sapidus, a well-studied model for crustacean osmoregulation. Overall, we confirmed previous expression patterns for individual ion transport genes and identified novel ones with salinity-mediated expression. Notable, novel DEGs among salinities and gill types for C. sapidus included anterior gills having higher expression of structural genes such as actin and cuticle proteins while posterior gills exhibit elevated expression of ion transport and energy-related genes, with the latter likely linked to ion transport. Potential targets among recovered DEGs for hormonal regulation of ion transport between salinities and gill types included neuropeptide Y and a KCTD16-like protein. Using publically available sequence data, constituents for a "core" gill transcriptome among decapods are presented, comprising genes involved in ion transport and energy conversion and consistent with salinity transfer experiments. Lastly, rarefication analyses lead us to recommend a modest number of sequence reads (~10-15M), but with increased biological replication, be utilized in future DEG analyses of crustaceans.

Influence of Bone and Muscle Injuries on the Osteogenic Potential of Muscle Progenitors: Contribution of Tissue Environment to Heterotopic Ossification.

UNLABELLED: : By using surgical mouse models, this study investigated how the tissue environment influences the osteogenic potential of muscle progenitors (m-progenitors) and potentially contributes to heterotopic ossification (HO). Injury was induced by clamping the gluteus maximus and medius (group M) or osteotomy of greater trochanter (group O) on the right hip, as well as combined muscle injury and osteotomy of greater trochanter (group M+O). The gluteus maximus and medius of the operated hips were harvested at days 1, 3, 5, and 10 for isolation of m-progenitors. The cells were cultured in an osteogenic medium for 3 weeks, and osteogenesis was evaluated by matrix mineralization and the expression of osteogenesis-related genes. The expression of type I collagen, RUNX2 (runt-related transcription factor 2), and osteocalcin by the m-progenitors of group M+O was significantly increased, compared with groups M and O. Osteogenic m-progenitors in group O increased the expression of bone morphogenetic protein 2 and also bone morphogenetic protein antagonist differential screening-selected gene aberrative in neuroblastoma. On histology, there was calcium deposition mostly in the muscles of group M+O harvested at day 10. CD56, representing myogenic progenitors, was highly expressed in the m-progenitors isolated from group M (day 10), but m-progenitors of group M+O (day 10) exhibited the highest expression of platelet-derived growth factor receptor α (PDGFR-α), a marker of muscle-derived mesenchymal stem cells (M-MSCs). The expressions of PDGFR-α and RUNX2 were colocalized in osteogenic m-progenitors. The data indicate that the tissue environment simulated in the M+O model is a favorable condition for HO formation. Most likely, M-MSCs, rather than myogenic progenitors, in the m-progenitors participate in HO formation. SIGNIFICANCE: The prevalence of traumatic heterotopic ossification (HO) is high in war injury. The pathogenesis of HO is still unknown. This study clarified the contribution of a tissue environment created by bone or muscle injury to the formation of HO. The study also found that muscle-derived mesenchymal stem cells, but not myogenic progenitors, are involved in the formation of HO. The findings of this study could be used to strategize the prevention and treatment of HO.

Pathological role of fibroblast-like synoviocytes in charcot neuroarthropathy.

This study was designed to characterize the synovium in the joints of Charcot neuroarthropathy (CNA) and investigate the potential role of fibroblast-like synoviocytes (FLS) in the pathology of CNA. Synovial samples were collected from CNA patients (n = 7) and non-CNA patients (n = 7), for control, during orthopaedic procedures and used for histology and isolation of FLS. Histological characterization of synovium included innervation and FLS localization. The isolated FLS from the CNA and non-CNA synovium were cultured, with or without tumor necrosis factor-α (TNF-α), for evaluation of invasiveness, gene expression, and cartilage degradation. Vasoactive intestinal peptide (VIP), a neuropeptide, was supplemented into the co-cultures of FLS and cartilage explants. Compared with the non-CNA synovium, CNA synovium was highly inflammatory, with reduced innervation and intense expression of cadherin-11. The FLS isolated from CNA synovium, particularly when activated with TNF-α, were more invasive, increased the expression of ADAMTS4, IL-6, and RANKL, and depleted proteoglycans from cartilage explants when they were co-cultured. Addition of VIP into the culture medium neutralized the catabolic effect of the CNA FLS on cartilage explants. In conclusion, FLS plays an important role in the pathology of CNA. Therapies targeting synovium and FLS may prevent or treat the joint destruction in CNA.

Implantation of Autologous Adipose Tissue-Derived Mesenchymal Stem Cells in Foot Fat Pad in Rats.

BACKGROUND: The foot fat pad (FFP) bears body weight and may become a source of foot pain during aging. This study investigated the regenerative effects of autologous adipose tissue-derived mesenchymal stem cells (AT-MSCs) in the FFP of rats. METHODS: Fat tissue was harvested from a total of 30 male Sprague-Dawley rats for isolation of AT-MSCs. The cells were cultured, adipogenic differentiation was induced for 1 week, and the AT-MSCs were labeled with fluorescent dye before injection. AT-MSCs (5 × 10(4) in 50 µL of saline) were injected into the second infradigital pad in the right hindfoot of the rat of origin. Saline only (50 µL) was injected into the corresponding fat pad in the left hind paw of each rat. Rats (n = 10) were euthanized at 1, 2, and 3 weeks, and the second infradigital fat pads were dissected for histologic examination. RESULTS: The fluorescence-labeled AT-MSCs were present in the foot pads throughout the 3-week experimental period. On histologic testing, the area of fat pad units (FPUs) in the fat pads that received AT-MSC injections was greater than that in the control fat pads. Although the thickness of septae was not changed by AT-MSC injections, the density of elastic fibers in the septae was increased in the fat pads with implanted AT-MSCs. CONCLUSION: In this short-term study, the implanted AT-MSCs largely survived and might have stimulated the expansion of individual FPUs and increased the density of elastic fibers in the FFP in this rat model. CLINICAL RELEVANCE: These data support the development of stem cell therapies for age-associated degeneration in FFP in humans.

Functional characterization of neuroendocrine regulation of branchial carbonic anhydrase induction in the euryhaline crab Callinectes sapidus.

Carbonic anhydrase (CA) plays an essential role as a provider of counterions for Na(+)/H(+) and Cl(-)/HCO3 (-) exchange in branchial ionic uptake processes in euryhaline crustaceans. CA activity and gene expression are low in crabs acclimated to full-strength seawater, with transfer to low salinity resulting in large-scale inductions of mRNA and subsequent enzyme activity in the posterior ion-regulating gills (e.g., G7). In the green crab Carcinus maenas, CA has been shown to be under inhibitory neuroendocrine control by a putative hormone in the x-organ-sinus gland complex (XOSG), located in the eyestalk. This study characterizes the neuroendocrine regulation of CA induction in the blue crab Callinectes sapidus, a commonly used experimental organism for crustacean osmoregulation. In crabs acclimated to full-strength seawater, eyestalk ligation (ESL) triggered a 1.8- and 100-fold increase in CA activity and mRNA, respectively. Re-injection with eyestalk homogenates abolished increases in CA activity and fractionally reduced CA gene expression. ESL also enhanced CA induction by 33% after 96 h in crabs transferred to 15 ppt salinity. Injection of eyestalk homogenates into intact crabs transferred from 35 to 15 ppt diminished by 43% the CA induction stimulated by low salinity. These results point to the presence of a repressor hormone in the eyestalk. Separate injections of medullary tissue (MT) and sinus gland (SG), two components of the eyestalk, reduced salinity-stimulated CA activity by 22% and 49%, suggesting that the putative repressor is localized to the SG. Crabs injected with SG extract harvested from crabs acclimated to 5 ppt showed no decrease in CA activity, demonstrating that the hormone is down-regulated at low salinity. Our results show the presence in the XOSG of an inhibitory compound that regulates salinity-stimulated CA induction.

Carbonic anhydrase induction in euryhaline crustaceans is rate-limited at the post-transcriptional level.

The transfer of euryhaline crustaceans from full-strength seawater to low salinity results in both a rapid up-regulation of carbonic anhydrase (CA; EC 4.2.1.1) mRNA and a slow induction of CA activity. There is a delay of several days between the two processes, which is attributed to the time required to synthesize new enzyme. These delays may also be due to limitations in the cellular uptake of Zn, which is a required post-translational active site modification to CA. To investigate these processes, the euryhaline crabs, Callinectes sapidus and Carcinus maenas, were acclimated to salinities below their isosmotic points (22.5 and 25 ppt, respectively) for 7 days to activate the physiological and molecular mechanisms of osmoregulation. CA mRNA increased 90-fold in C. sapidus and 2-fold in C. maenas within 6h; whereas it took 48 h for the initial increases in CA activity (120% and 31%), and 4 to 7 days for new acclimated levels (300% and 100%, respectively). Crabs were then transferred to lower salinities (10 and 15 ppt) to induce further CA activity and to determine if previous increases in CA mRNA reduced the time required for subsequent CA induction. Additionally, the expression of the Zn transporter ZIP1 was examined in C. sapidus at 35 and 22.5 ppt. In both species, prior CA mRNA elevation failed to accelerate the rate of CA induction. Levels of CA mRNA did not change in either crab following transfer from intermediate to low salinity. Taken together, these results show that the timecourse of CA induction at low salinity is not limited by the expression of CA mRNA, but by the synthesis of new enzyme from an existing pool of mRNA. No increases in ZIP1 expression occurred at low salinity, therefore these delays may be due to the limits of cellular Zn uptake.