Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

The effect of X-ray micro computed tomography image resolution on flow properties of porous rocks.

The study of digital rock physics has seen significant advances due to the development of X-ray micro computed tomography scanning devices. One of the advantages of using such a device is that the pore structure of rock can be mapped down to the micrometre level in three dimensions. However, in providing such high-resolution images (low voxel size), the resulting file sizes are necessarily large (of the order of gigabytes). Lower image resolution (high voxel size) produces smaller file sizes (of the order of hundreds of megabytes), but risks losing significant details. This study describes the effect of the image resolution obtained by means of hardware-based and software-based approach. Four samples of porous rock were scanned using a SkyScan 1173 High Energy Micro-CT. We found that acquisition using increased pixel binning of the camera (hardware-based resizing) significantly affects the calculated physical properties of the samples. By contrast, voxel resizing by means of a software-based approach during the reconstruction process yielded less effect on the porosity and specific surface area of the samples. However, the decreasing resolution of the image obtained by both the hardware-based and the software-based approaches affects the permeability significantly. We conclude that simulating fluid flow through the pore space using the Lattice Boltzmann method to calculate the permeability has a significant dependency on the image resolution.

Image analysis in medical imaging: recent advances in selected examples.

Medical imaging has developed into one of the most important fields within scientific imaging due to the rapid and continuing progress in computerised medical image visualisation and advances in analysis methods and computer-aided diagnosis. Several research applications are selected to illustrate the advances in image analysis algorithms and visualisation. Recent results, including previously unpublished data, are presented to illustrate the challenges and ongoing developments.

Altered interactions of tryptophan metabolites in first-episode neuroleptic-naive patients with schizophrenia.

Schizophrenia is characterized by complex and dynamically interacting perturbations in multiple neurochemical systems. In the past, evidence for these alterations has been collected piecemeal, limiting our understanding of the interactions among relevant biological systems. Earlier, both hyper- and hyposerotonemia were variously associated with the longitudinal course of schizophrenia, suggesting a disturbance in the central serotonin (5-hydroxytryptamine (5-HT)) function. Using a targeted electrochemistry-based metabolomics platform, we compared metabolic signatures consisting of 13 plasma tryptophan (Trp) metabolites simultaneously between first-episode neuroleptic-naive patients with schizophrenia (FENNS, n=25) and healthy controls (HC, n=30). We also compared these metabolites between FENNS at baseline (BL) and 4 weeks (4w) after antipsychotic treatment. N-acetylserotonin was increased in FENNS-BL compared with HC (P=0.0077, which remained nearly significant after Bonferroni correction). N-acetylserotonin/Trp and melatonin (Mel)/serotonin ratios were higher, and Mel/N-acetylserotonin ratio was lower in FENNS-BL (all P-values<0.0029), but not after treatment, compared with HC volunteers. All three groups had highly significant correlations between Trp and its metabolites, Mel, kynurenine, 3-hydroxykynurenine and tryptamine. However, in the HC, but in neither of the FENNS groups, serotonin was highly correlated with Trp, Mel, kynurenine or tryptamine, and 5-hydroxyindoleacetic acid (5HIAA) was highly correlated with Trp, Mel, kynurenine or 3-hydroxykynurenine. A significant difference between HC and FENNS-BL was further shown only for the Trp-5HIAA correlation. Thus, some metabolite interactions within the Trp pathway seem to be altered in the FENNS-BL patients. Conversion of serotonin to N-acetylserotonin by serotonin N-acetyltransferase may be upregulated in FENNS patients, possibly related to the observed alteration in Trp-5HIAA correlation. Considering N-acetylserotonin as a potent antioxidant, such increases in N-acetylserotonin might be a compensatory response to increased oxidative stress, implicated in the pathogenesis of schizophrenia.

Exploiting the tumor microenvironment in the development of targeted cancer gene therapy.

The future success of cancer gene therapy is critically dependent upon the development of safe, practical and effective targeting strategies. In this study, we describe a novel and broadly applicable targeting approach in which the induction of apoptotic tumor cell death is linked to the differential expression within the tumor microenvironment of elevated levels of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF). As VEGF is generally absent or produced at only low levels in most normal tissues, undesirable toxicity will not result even if the therapeutic gene in question is inadvertently expressed in non-targeted tissue sites. The basic approach makes use of a chimeric cell-surface protein in which the membrane-spanning and cytoplasmic 'death domain' of the pro-apoptotic protein Fas are fused in frame to the extracellular ligand-binding domain of the VEGF receptor Flk-1/KDR/VEGFR2 (Flk-1/Fas). The resultant chimeric Flk-1/Fas receptor was found to be stable and capable of inducing a rapid apoptotic response when expressed in tumor cells that produce endogenous VEGF. Importantly, in the absence of VEGF, transduced tumor cells remain viable although they can be triggered to die by the addition of recombinant VEGF. Given the key role played by VEGF in tumor development and progression, it is proposed that the Flk-1/Fas chimera may have great potential in the context of tumor cell-targeted cancer gene therapy.

Fractal signature and lacunarity in the measurement of the texture of trabecular bone in clinical CT images.

Fractal analysis is a method of characterizing complex shapes such as the trabecular structure of bone. Numerous algorithms for estimating fractal dimension have been described, but the Fourier power spectrum method is particularly applicable to self-affine fractals, and facilitates corrections for the effects of noise and blurring in an image. We found that it provided accurate estimates of fractal dimension for synthesized fractal images. For natural texture images fractality is limited to a range of scales, and the fractal dimension as a function of spatial frequency presents as a fractal signature. We found that the fractal signature was more successful at discriminating between these textures than either the global fractal dimension or other metrics such as the mean width and root-mean-square width of the spectral density plots. Different natural textures were also readily distinguishable using lacunarity plots, which explicitly characterize the average size and spatial organization of structural sub-units within an image. The fractal signatures of small regions of interest (32x32 pixels), computed in the frequency domain after corrections for imaging system noise and MTF, were able to characterize the texture of vertebral trabecular bone in CT images. Even small differences in texture due to acquisition slice thickness resulted in measurably different fractal signatures. These differences were also readily apparent in lacunarity plots, which indicated that a slice thickness of 1 mm or less is necessary if essential architectural information is not to be lost. Since lacunarity measures gap size and is not predicated on fractality, it may be particularly useful for characterizing the texture of trabecular bone.

A comparison of the texture of computed tomography and projection radiography images of vertebral trabecular bone using fractal signature and lacunarity.

The structural integrity of trabecular bone is an important factor characterizing the biomechanical strength of the vertebra, and is determined by the connectivity of the bone network and the trabeculation pattern. These can be assessed using texture measures such as the fractal signature and lacunarity from a high resolution projection radiograph. Using central sections of lumbar vertebrae we compared the results obtained from high-resolution transverse projection images with those obtained from spatially registered low-resolution images from a conventional clinical CT scanner to determine whether clinical CT data can provide useful structural information. Provided the power spectra of the CT images are corrected for image system blurring, the resulting fractal signature is similar for both modalities. Although the CT images are blurred relative to the projection images, with a consequent reduction in lacunarity, the estimated trabecular separation obtained from the lacunarity plots is similar for both modalities. This suggests that these texture measures contain essential information on trabecular microarchitecture, which is present even in low resolution CT images. Such quantitative texture measurements from CT or MRI images are potentially useful in monitoring bone strength and predicting future fracture risk.

Bone density measured by dual-energy X-ray absorptiometry in healthy Kuwaiti women.

A preliminary cross-sectional study of 623 healthy Kuwaiti women aged 20-79 years was carried out using dual-energy X-ray absorptiometry (DXA) to establish reference values of bone mineral density (BMD). Measurements were taken at the lumbar spine and proximal femur. The data were compared with normative data taken from Caucasian U.S./North European women over 6 decades of age. Our results showed the expected decline in BMD with age, especially for postmenopausal women. Despite differences in ethnicity, diet, and lifestyle no statistically significant differences in BMD were detected between the Kuwaiti subjects and the Caucasian normative data. Age-matched, clinically well-defined normative data for a given population must be used to diagnose osteoporosis and establish fracture risk. This study represents baseline data for Kuwaiti Arab females: it indicates that their BMD values are close to those of Caucasian females and higher than those reported recently for Lebanese and Saudi females. Further data is being collected to increase the statistical power of the study.

Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44.

Elevated tumor cyclooxygenase (COX-2) expression is associated with increased angiogenesis, tumor invasion, and suppression of host immunity. We have previously shown that genetic inhibition of tumor COX-2 expression reverses the immunosuppression induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, NSCLC cell lines were transduced with a retroviral vector expressing the human COX-2 cDNA in the sense (COX-2-S) and antisense (COX-2-AS) orientations. COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more prostaglandin E(2), and demonstrated an enhanced invasive capacity compared with control vector-transduced or parental cells. CD44, the cell surface receptor for hyaluronate, was overexpressed in COX-2-S cells, and specific blockade of CD44 significantly decreased tumor cell invasion. In contrast, COX-2-AS clones had a very limited capacity for invasion and showed diminished expression of CD44. These findings suggest that a COX-2-mediated, CD44-dependent pathway is operative in NSCLC invasion. Because tumor COX-2 expression appears to have a multifaceted role in conferring the malignant phenotype, COX-2 may be an important target for gene or pharmacologic therapy in NSCLC.

Mechanisms mediating the effects of IL-3 gene expression on tumor growth.

IL-3 gene expression within tumors leads to host-cell infiltration, particularly by macrophages, slower tumor growth, and enhanced immunogenicity. Surprisingly, tumor-associated macrophages (TAMs) from within FSAN-JmIL3 tumors had decreased expression of TNF-alpha and iNOS. On short-term culture, TAMs from FSAN-JmIL3 tumors regained their capacity to produce TNF-alpha and NO, indicating that they were primed in vivo. In vitro experiments were unable to demonstrate differences between FSAN-JmIL3 and FSAN tumor cells in their ability to stimulate TNF-alpha production by TAMs. In the absence of evidence that TAM activation was responsible for the slower growth of FSAN-JmIL3 tumors, the response of tumor cells to these effector molecules was studied. TNF-alpha and NO were cytotoxic for FSAN-JmIL3 cells but growth stimulatory for FSAN. These tumor-related phenotypic changes may contribute as much if not more than functional changes in host infiltrating cells to the slower growth of FSAN-JmIL3 tumors in vivo.

Combining radiation therapy with interleukin-3 gene immunotherapy.

The goal of this study was to explore immunological strategies to increase local and systemic tumor control in patients receiving radiation therapy. In previous studies, interleukin-3 (IL-3) gene expression within murine tumors was shown to increase their response to irradiation through immune mechanisms. In this study, the efficacy of systemically administered IL-3 gene-transduced irradiated tumor cell vaccines was tested for their ability to augment radiation responses against established immunogenic (FSAR) and nonimmunogenic (FSAN) tumors. Vaccines of irradiated FSAR/FSAN or FSAN-JmIL-3/FSAR-JmIL-3 cells were given intraperitoneally just before and after local irradiation of parental tumors with diameters of 8 mm, as well as in two booster doses. The IL-3 gene-transduced tumor cell vaccines were more effective than the parental vaccines at delaying tumor growth after irradiation, although no complete cures resulted. Responses were largely specific to the tumor type, indicating that tumor-specific immunity was enhanced by IL-3 vaccine administration. When the experiment was repeated in the C3H/HeJ mice, which are deficient in tumor necrosis factor-alpha production, the vaccines were still effective, but less so than in C3H/HeN mice. Systemic IL-3 vaccine treatment increased intratumoral levels of intercellular adhesion molecule-1, Mac-1, EB22/5.3, tumor necrosis factor-alpha, and IL-1 mRNA in irradiated tumors, indicating that cellular infiltration was part of the response. The study demonstrates that local radiation therapy can enhance the efficacy of genetically altered vaccine-based immunotherapy for cancer by decreasing tumor burden. At the same time, tumor cell vaccines may improve the cure rate of local radiation therapy by eliminating residual cancer cells. Although less effective than intratumoral gene expression, administration of IL-3 gene-transduced tumor cell vaccines is clinically a more feasible strategy that may be useful in situations in which the tumor load is small.

Ectopic expression of Rsu-1 results in elevation of p21CIP and inhibits anchorage-independent growth of MCF7 breast cancer cells.

Signal transduction from tyrosine kinase receptors mediates growth regulation of breast cancer cells in part through the GTPase Ras and downstream kinases. Rsu-1 is a cDNA previously identified as an inhibitor of Ras-induced transformation. An HA-epitope tagged Rsu-1 cDNA was introduced into the MCF7 breast carcinoma cell line. Stable transfectants were selected and used for analysis of Rsu-1 expression on growth control and Ras-dependent kinase pathways. Assessment of biological activity of HA-Rsu-1 transfectants revealed that HA-Rsu-1 clones showed slower anchorage dependent growth rates than control MCF7 cell lines and a significant reduction in anchorage independent growth. Analysis of cell cycle regulatory proteins required for transit through G1 revealed that HA-Rsu-1 transfectant cell lines expressed elevated levels of p21CIP CDK inhibitor. Perturbations in signal transduction pathways which can be activated by Ras were detected in the Ha-Rsu-1 transfectants. Exposure of serum-starved cells to EGF revealed that expression of HA-Rsu-1 increased ERK-2 kinase activation, decreased activation of Jun kinase and inhibited Rho-dependent Rho-alpha kinase (ROK) activity compared to control cells. While serum starvation reduced AKT activity to undetectable levels in HA-Rsu-1 transfectants but not in control MCF7 cells, activation of AKT kinase by serum was unaffected by HA-Rsu-1 expression. Finally, the level of c-myc transcription in HA-Rsu-1 transfectants reached only 60% of the MCF7 control cell line following serum stimulation of starved cells while Fos RNA levels were similar to control cells. These results demonstrate that increased Rsu-1 expression critically altered cell cycle regulation and growth of MCF7 cells as well as signaling pathways in MCF7 cells required for malignant growth.

A quantitative index for the measurement of the tortuosity of blood vessels.

We propose a novel index for quantitating arterial tortuosity, based on second differences of the coordinates of the vessel midline. The index is independent of the magnification of the image and of sampling frequency. We have demonstrated its validity as an indicator of changes in morphology using simulated shapes. It is superior to two other putative indices based on vessel elongation and the variance of differences in the vessel midline coordinates, presented previously in the literature. The proposed index is easily generalised to three dimensions, allowing a true three-dimensional tortuosity index to be calculated from biplane angiograms or serial tomographic slices. It has a number of clinical applications. Preliminary data, using 82 aortograms from radiographic archives, showed a significant correlation between the tortuosity of the abdominal aorta and subject age (r=0.684, P<0.001). For this group, increased tortuosity was especially evident for subjects above 40 yr of age.

The peak CT number of profiles perpendicular to the vertebral cortical shell may be a useful indicator of the integrity of the cortical shell.

Limited spatial resolution affects the ability of clinical CT systems to directly measure the thickness and density of the vertebral cortical shell. In such cases the peak CT number (CTp) of a profile through the cortical shell is approximately proportional to the product of thickness and density and hence an average of several of these values (CTp) may be useful as an indicator of the integrity of the cortical shell. Values of CTp were determined around the anterior two-thirds of the cortical shell of the L3 and L4 lumbar vertebrae of 96 Kuwaiti women (mean age+/-SD=50.0+/-8.5). There was no significant difference between the two vertebrae in terms of CTp and trabecular BMD values. There was a significant correlation between CTp and trabecular BMD for each vertebra (r=0.597, p<0.0001), and between CTp and patient age (r=0.624, p<0.001), suggesting that both cortical and trabecular bone may be subject to similar thinning mechanisms during ageing and osteoporosis. The variation of CTp around the cortical shell, represented by the standard deviation of nine CTp measurements, was significantly correlated with the mean value (r=0.503, p<0.0001) and may be useful as a descriptor of cortical structure.

Effect of sub-pixel misregistration on the determination of the point spread function of a CT imaging system.

The point spread function (PSF) of an imaging system provides a complete, quantitative description of its resolution. In computed tomography (CT) it can be obtained conveniently and directly from the image of a thin metal wire. However, the shape of the observed PSF is affected by arbitrary sub-pixel shifts in alignment between the wire object and the imaging raster, causing partial volume averaging. We investigated the loss of symmetry of the PSF due to this misregistration, using a thin wire at a small angle to the axis of the CT scanner. We were able to identify the optimal registration, and hence the best estimate of the PSF, from the minimum skewness of a series of images. Determination of an exact PSF is important in the imaging of structures of sub-pixel size, such as small blood vessels or trabeculae in spongy bone. The method is particularly useful for modalities with coarse rasters such as magnetic resonance imaging and nuclear medicine.

Measurement of thickness and density of thin structures by computed tomography: a simulation study.

The limited spatial resolution of clinical CT systems causes difficulties in the measurement of the density and thickness of thin structures such as the vertebral cortical shell. We simulated the imaging process by convolving experimentally determined point spread functions with rectangular and Gaussian profiles, for various fields of view or pixel sizes and reconstruction kernels. The simulations successfully explained the reported overestimation of thickness and underestimation of density when imaging thin structures. Both effects are larger for Gaussian profiles. For the rectangular profiles, experimental estimates of thickness and density will only be accurate when the true thickness is greater than about 1.5 times (for the bone reconstruction kernel) or 2.0 times (for the standard kernel) the full width at half maximum of the point spread function (PSF) of the imaging system. For Gaussian profiles imaged by a system with a Gaussian PSF, there are straightforward analytical expressions for the overestimation of thickness and underestimation of density: and these are useful approximations to the simulations of Gaussian profiles with experimental (pseudo-Gaussian) PSFs. We have demonstrated that thresholding of the vertebral image cannot provide accurate estimates of cortical thickness and density because the appropriate threshold level requires foreknowledge of the cortical thickness. To circumvent such difficulties we suggest that the average value of the peak CT numbers measured along the medial axis of the cortical shell be adopted as an index of cortical shell strength, since its value depends on both the density and the thickness of the shell.

Newly discovered anti-inflammatory properties of the benzamides and nicotinamides.

Our laboratory has concentrated on the possible regulation the benzamides and nicotinamides may have on the processes of DNA repair and apoptosis. Recent reports have suggested that both apoptosis and inflammation are regulated by the transcription factor NF-kappaB. We have initiated studies regarding the hypothesis that the benzamides and nicotinamides could inhibit the production of tumor necrosis factor alpha (TNFalpha) and the inflammatory response as well as induce apoptosis via inhibition of NF-kappaB. Our data have shown that nicotinamide and two N-substituted benzamides, metoclopramide (MCA) and 3-chloroprocainamide (3-CPA), gave dose dependent inhibition of lipopolysacharide induced TNFalpha in the mouse within the dose range of 10-500 mg/kg. Moreover, lung edema was prevented in the rat by 3 x 50 mg/kg doses of 3-CPA or MCA, and 100-200 microM doses of MCA could also inhibit NF-kappaB in Hela cells. Taken together these data strongly support the notion that benzamides and nicotinamides have potent anti-inflammatory and antitumor properties, because their primary mechanism of action is regulated by inhibition at the gene transcription level of NF-kappaB, which in turn inhibits TNFalpha and induces apoptosis.

Photodynamic therapy-mediated immune response against subcutaneous mouse tumors.

The curative ability of photodynamic therapy (PDT) is severely compromised if treated tumors are growing in immunodeficient hosts. Reconstitution of severe combined immunodeficient (scid) mice with splenocytes from naive immunologically intact BALB/c mice did not improve the response to Photofrin-based PDT of EMT6 tumors growing in these animals. In contrast, adoptive transfer of BALB/c splenocytes containing EMT6 tumor-sensitized immune cells had a dramatic effect on tumor regrowth after PDT. For instance, full restoration of the curative effect of PDT was achieved with scid mice that received splenocytes from BALB/c donors that were cured of EMT6 tumors by PDT 5 weeks before adoptive transfer. Splenocytes obtained from donors cured of EMT6 tumors using X-rays were much less effective. Selective in vitro depletion of specific T-cell populations from engrafting splenocytes indicated that CTLs are the main immune effector cells responsible for conferring the curative outcome to PDT in this experimental model, whereas helper T lymphocytes play a supportive role. The immune specificity of these T-cell populations was demonstrated by the absence of cross-reactivity between the EMT6 and Meth-A tumor models (mismatch between tumors growing in splenocyte donors and recipients). The immunocompetent BALB/c mice that received adoptively transferred splenocytes containing PDT-generated, tumor-sensitized immune cells also benefited from the improved outcome of PDT of tumors they were bearing. This was demonstrated not only with the fairly immunogenic EMT6 tumor model but also with weakly immunogenic Line 1 carcinomas. The results of this study indicate that PDT is a highly effective means of generating tumor-sensitized immune cells that can be recovered from lymphoid sites distant to the treated tumor at protracted time intervals after PDT, which asserts their immune memory character. It is also shown that the treatment of tumors by PDT creates the conditions necessary for converting the inactive adoptively transferred pre-effector, tumor-sensitized immune cells into fully functional antitumor effector cells. An additional finding of this study is the evidence of NK cell activation in PDT-treated Meth-A sarcomas.