The effect of dimensionality on growth and differentiation of neural progenitors from different regions of fetal rat brain in vitro: 3-dimensional spheroid versus 2-dimensional monolayer culture

Three-dimensional (3-D) spheroids are widely used for culturing cells. However, 2-dimensional (2-D) monolayer cultures have also been adopted for culture and used in a broad range of cell biology studies. To address the effect of dimensionality on the growth and differentiation of neuroprogenitor cells in 3-D spheroids and 2-D monolayer cultures, cells were isolated from cerebral cortex, cerebella and brainstem of fetal rat brain then cultured in serum-free DMEM/F12 medium or DMEM with 10% FBS. The growth and differentiation of neuroprogenitor cells from three brain regions in spheroids was compared with that in monolayer cultures, and the differentiation components of neuroprogenitor cells were compared with in vivo brain sections. Neuroprogenitor cells in spheroids proliferate actively over 10 days in culture as showed by Ki67 incorporation and increase in spheroid diameter. More neuroprogenitor cells underwent neuronal differentiation in spheroids than in monolayer cultures. In comparison with fixed rat brain sections, the neuron to astrocyte ratio, as shown by neurofilament to glial fibrillary acidic protein immunoreactivity, in spheroids is similar to that found in adult rat tissue sections. Our results suggest that the spheroid culture system mimics the in vivo cytoarchitecture to a greater extent and more closely reflects the cellular composition in adult brain tissue. This supports the notion that the intercellular niche in spheroids is more favorable for the survival and differentiation of neuronal precursors, while the cues in monolayer cultures may favor glial cell survival. It is therefore concluded that dimensionality plays a significant role in determining cellular behavior in vitro.     

Population‐based screening for Lynch syndrome in Western Australia

We showed earlier that routine screening for microsatellite instability (MSI) and loss of mismatch repair (MMR) protein expression in colorectal cancer (CRC) led to the identification of previously unrecognized cases of Lynch syndrome (LS). We report here the results of screening for LS in Western Australia (WA) during 1994–2012. Immunohistochemistry (IHC) for loss of MMR protein expression was performed in routine pathology laboratories, while MSI was detected in a reference molecular pathology laboratory. Information on germline mutations in MMR genes was obtained from the state's single familial cancer registry. Prior to the introduction of routine laboratory‐based screening, an average of 2–3 cases of LS were diagnosed each year amongst WA CRC patients. Following the implementation of IHC and/or MSI screening for all younger (<60 years) CRC patients, this has increased to an average of 8 LS cases diagnosed annually. Based on our experience in WA, we propose three key elements for successful population‐based screening of LS. First, for all younger CRC patients, reflex IHC testing should be carried out in accredited pathology services with ongoing quality control. Second, a state‐ or region‐wide reference laboratory for MSI testing should be established to confirm abnormal or suspicious IHC test results and to exclude sporadic cases by carrying out BRAF mutation or MLH1 methylation testing. Finally, a state or regional LS coordinator is essential to ensure that all appropriate cases identified by laboratory testing are referred to and attend a Familial Cancer Clinic for follow‐up and germline testing.     

Ipilimumab retreatment in patients with pretreated advanced melanoma: the expanded access programme in Italy

Background:

Retreatment with ipilimumab has been shown to re-establish disease control in some patients with disease progression. Here, we report the efficacy and safety of retreatment with ipilimumab 3 mg kg⁻¹ among patients participating in an expanded access programme in Italy.

Methods:

Patients who achieved disease control during induction therapy were retreated with ipilimumab upon progression (3 mg kg⁻¹ every 3 weeks for up to four doses), providing they had not experienced toxicity that precluded further dosing. Tumour assessments were conducted after retreatment, and patients were monitored throughout for adverse events.

Results:

Of 855 patients treated with ipilimumab, 51 were retreated upon disease progression. Of these, 28 (55%) regained disease control upon retreatment and 42% were alive 2 years after the first induction dose of ipilimumab; median overall survival was 21 months. Eleven patients (22%) had a treatment-related adverse event of any grade during retreatment. These were generally mild-to-moderate and resolved within a median of 4 days. No new types of toxicity were reported.

Conclusions:

For patients who meet predefined criteria, retreatment with ipilimumab is generally well tolerated and can translate into clinical benefit. This strategy should be compared with other therapeutic options in randomised controlled trials.     

Work-Related Injuries Among Immigrant Workers in Italy

In Italy, work-related injuries among immigrant workers are an emerging concern. In this study, we compared the occurrence of work-related injuries between legally residing immigrants from High Migration Pressure Countries and Italians and evaluated the associations with potential risk factors. Using data from the 2007 Labour Force Survey conducted by Italy’s National Institute of Statistics, we examined the relationship between the occurrence of work-related injuries in the previous 12 months and being an immigrant among a nationally representative sample. The occurrence of work-related injuries was significantly higher among immigrant males compared to Italian males (adjusted OR = 1.82; 95 % CI 1.53–2.16), particularly in the construction sector, for which the results showed a U-shaped trend of the odds ratios of injuries for immigrants compared to Italians with increasing number of years of work in the same job. No associations were found among women. The findings suggest that prevention programs need to be implemented to limit the burden of work-related injuries among immigrants.     

Type-1 cannabinoid receptors colocalize with caveolin-1 in neuronal cells

Type-1 (CB1) and type-2 (CB2) cannabinoid receptors belong to the rhodopsin family of G protein-coupled receptors, and are activated by endogenous lipids termed "endocannabinoids". Recent reports have demonstrated that CB1R, unlike CB2R and other receptors and metabolic enzymes of endocannabinoids, functions in the context of lipid rafts, i.e. plasma membrane microdomains which may be important in modulating signal transduction. Here, we present novel data based on cell subfractionation, immunoprecipitation and confocal microscopy studies, that show that in C6 cells CB1R co-localizes almost entirely with caveolin-1. We also show that trafficking of CB1R in response to the raft disruptor methyl-beta-cyclodextrin (MCD) is superimposable on that of caveolin-1, and that MCD treatment increases the accessibility of CB1R to its specific antibodies. These findings may be relevant for the manifold CB1R-dependent activities of endocannabinoids, like the regulation of apoptosis and of neurodegenerative diseases.     

Vertebral synovial osteochondromatosis with compressive myelopathy.

STUDY DESIGN: A case report of vertebral synovial osteochondromatosis with compressive myelopathy. OBJECTIVES: To describe the clinical, radiologic, and histopathologic features of vertebral facet synovial osteochondromatosis with compressive myelopathy. SUMMARY OF BACKGROUND DATA: There has been only one previously reported case of synovial osteochondromatosis affecting the vertebral facet joint and no previous report of associated compressive myelopathy. METHODS: The case history, radiology, surgical findings, and histopathology are reviewed. RESULTS: Vertebral facet synovial osteochondromatosis is a potential and readily manageable cause of spinal cord compression. CONCLUSIONS: Synovial osteochondromatosis of the vertebral facet joint should be considered as a cause of compressive myelopathy.     

Pharmacological management of IPF

Idiopathic pulmonary fibrosis (IPF) is a deadly disease with a median survival of approximately three years in historical cohorts. Despite increased knowledge of disease pathophysiology and selection of more targeted therapy, main clinical trials yielded negative results. However, two agents, pirfenidone and nintedanib, were recently shown to be effective in IPF and received marketing authorization worldwide. Both drugs significantly reduce functional decline and disease progression with an acceptable safety profile. Yet, none of these drugs actually improves or even stabilizes the disease or the symptoms perceived by the patient. Several other treatments and combinations are currently tested, and many more are ready for clinical trials. Their completion is critical for achieving the ultimate goal of curing patients with IPF.     

CD4+CD25+ T cells alloactivated ex vivo by IL-2 or IL-4 become potent alloantigen-specific inhibitors of rejection with different phenotypes, suggesting separate pathways of activation by Th1 and Th2 responses

CD4(+)CD25(+)Foxp3(+) T cells are regulatory/suppressor cells (Tregs) that include non-antigen (Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naive CD4(+)CD25(+) Treg develop into specific Tregs is unknown. Here, we generated adaptive Tregs by culture of naive CD4(+)CD25(+)Foxp3(+) T cells with allo-Ag and either interleukin-2 (IL-2) or IL-4. Within days, IL-2 enhanced interferon-gamma receptor (Ifngammar) and Il-5 mRNA and IL-4 induced a reciprocal profile with de novo IL-5Ralpha and increased IFN-gamma mRNA expression. Both IL-2- and IL-4-alloactivated CD4(+)CD25(+) Tregs within 3 to 4 days of culture had enhanced capacity to induce tolerance to specific donor but not to third-party cardiac allografts. These hosts became tolerant as allografts functioned more than 250 days, with a physiologic ratio of less than 10% CD4(+)CD25(+)Foxp3(+) T cells in the CD4(+) population. CD4(+)CD25(+) T cells from tolerant hosts given IL-2-cultured cells had increased Il-5 and Ifngammar mRNA. Those from hosts given IL-4-cultured cells had enhanced IL-5Ralpha mRNA expression and IL-5 enhanced their proliferation to donor but not third-party allo-Ag. Thus, IL-2 and IL-4 activated allo-Ag-specific Tregs with distinct phenotypes that were retained in vivo. These findings suggested that T-helper 1 (Th1) and Th2 responses activate 2 pathways of adaptive Ag-specific Tregs that mediate tolerance. We propose they be known as T-suppressor 1 (Ts1) and Ts2 cells.     

Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance

The ESCRT pathway helps mediate the final abscission step of cytokinesis in mammals and archaea. In mammals, two early acting proteins of the ESCRT pathway, ALIX and TSG101, are recruited to the midbody through direct interactions with the phosphoprotein CEP55. CEP55 resides at the centrosome through most of the cell cycle but then migrates to the midbody at the start of cytokinesis, suggesting that the ESCRT pathway may also have centrosomal links. Here, we have systematically analyzed the requirements for late-acting mammalian ESCRT-III and VPS4 proteins at different stages of mitosis and cell division. We found that depletion of VPS4A, VPS4B, or any of the 11 different human ESCRT-III (CHMP) proteins inhibited abscission. Remarkably, depletion of individual ESCRT-III and VPS4 proteins also altered centrosome and spindle pole numbers, producing multipolar spindles (most ESCRT-III/VPS4 proteins) or monopolar spindles (CHMP2A or CHMP5) and causing defects in chromosome segregation and nuclear morphology. VPS4 proteins concentrated at spindle poles during mitosis and then at midbodies during cytokinesis, implying that these proteins function directly at both sites. We conclude that ESCRT-III/VPS4 proteins function at centrosomes to help regulate their maintenance or proliferation and then at midbodies during abscission, thereby helping ensure the ordered progression through the different stages of cell division.