Survey on Transfusion-Transmitted Cytomegalovirus and Cytomegalovirus Disease Mitigation.

CONTEXT: - Cytomegalovirus (CMV) can be transmitted by cellular blood products, leading to severe disease in immunosuppressed patients such as neonates and transplant recipients. To mitigate transfusion-transmitted CMV (TT-CMV), "CMV-safe" blood products (leukoreduced and/or CMV-seronegative) are transfused. Attempts to develop practice guidelines for TT-CMV mitigation have been limited by paucity of high-quality clinical trials. OBJECTIVE: - To assess current TT-CMV mitigation strategies across medical institutions for specific at-risk populations. DESIGN: - Supplemental questions regarding TT-CMV and CMV disease mitigation were added to a College of American Pathologists Transfusion Medicine (Comprehensive) Participant Survey in 2015, addressing whether a given institution provided CMV-safe products for 6 at-risk patient populations. RESULTS: - Ninety percent (2712 of 3032) of institutions reported providing universally leukoreduced blood products. Among institutions without universal leukoreduction, 92% (295 of 320) provided leukoreduced products on the basis of clinical criteria. Eighty-three percent (2481 of 3004) of respondents reported having availability of CMV-seronegative products; however, wide variation in policies was reported governing CMV-seronegative product use. Among all respondents, less than 5% reported using CMV prophylaxis and monitoring in high-risk patient groups. Transplant centers reported higher rates of CMV prophylaxis (25% [97 of 394] solid organ) and monitoring (15% [59 of 394] solid organ) for CMV-negative transplant recipients. CONCLUSIONS: - Universal leukoreduction is the primary strategy for mitigating TT-CMV. While most institutions have both CMV-seronegative and leukoreduced blood products available, consensus is lacking on which patients should receive these products. High-quality studies are needed to determine if CMV-seronegative and leukoreduced blood products are needed in high-risk patient populations.

Adjudin--A Male Contraceptive with Other Biological Activities.

BACKGROUND: Adjudin has been explored as a male contraceptive for the last 15 years since its initial synthesis in the late 1990s. More than 50 papers have been published and listed in PubMed in which its mechanism that induces exfoliation of germ cells from the seminiferous epithelium, such as its effects on actin microfilaments at the apical ES (ectoplasmic specialization, a testis-specific actin-rich anchoring junction) has been delineated. OBJECTIVE: Recent studies have demonstrated that, besides its activity to induce germ cell exfoliation from the seminiferous epithelium to cause reversible infertility in male rodents, adjudin possesses other biological activities, which include anti-cancer, anti-inflammation in the brain, and anti-ototoxicity induced by gentamicin in rodents. Results of these findings likely spark the interest of investigators to explore other medical use of this and other indazole-based compounds, possibly mediated by the signaling pathway(s) in the mitochondria of mammalian cells following treatment with adjudin. In this review, we carefully evaluate these recent findings. METHODS: Papers published and listed at www.pubmed.org and patents pertinent to adjudin and its related compounds were searched. Findings were reviewed and critically evaluated, and summarized herein. RESULTS: Adjudin is a novel compound that possesses anti-spermatogenetic activity. Furthermore, it possesses anti-cancer, anti-inflammation, anti-neurodegeneration, and anti-ototoxicity activities based on studies using different in vitro and in vivo models. CONCLUSION: Studies on adjudin should be expanded to better understand its biological activities so that it can become a useful drug for treatment of other ailments besides serving as a male contraceptive.

Role of non-receptor protein tyrosine kinases in spermatid transport during spermatogenesis.

Non-receptor protein tyrosine kinases are cytoplasmic kinases that activate proteins by phosphorylating tyrosine residues, which in turn affect multiple functions in eukaryotic cells. Herein, we focus on the role of non-receptor protein tyrosine kinases, most notably, FAK, c-Yes and c-Src, in the transport of spermatids across the seminiferous epithelium during spermatogenesis. Since spermatids, which are formed from spermatocytes via meiosis, are immotile haploid cells, they must be transported by Sertoli cells across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Without the timely transport of spermatids across the epithelium, the release of sperms at spermiation fails to occur, leading to infertility. Thus, the molecular event pertinent to spermatid transport is crucial to spermatogenesis. We provide a critical discussion based on recent findings in this review. We also provide a hypothetical model on spermatid transport, and the role of non-receptor protein tyrosine kinases in this event. We also highlight areas of research that deserve attention by investigators in the field.

Actin binding proteins, spermatid transport and spermiation.

The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.

Interaction of oligomeric breast cancer resistant protein (BCRP) with adjudin: a male contraceptive with anti-cancer activity.

Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein foun d in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis.

RAI14 (retinoic acid induced protein 14) is an F-actin regulator: Lesson from the testis.

RAI14 (retinoic acid induced protein 14) is an actin-binding protein first identified in the liver. In the testis, RAI14 is expressed by both Sertoli and germ cells in the seminiferous epithelium. Besides binding to actin in the testis, RAI14 is also a binding protein for palladin, an actin cross-linking and bundling protein. A recent report has shown that RAI14 displays stage-specific and spatiotemporal expression at the ES [ectoplasmic specialization, a testis-specific filamentous (F)-actin-rich adherens junction] in the seminiferous epithelium of adult rat testes during the epithelial cycle of spermatogenesis, illustrating its likely involvement in F-actin organization at the ES. Functional studies in which RAI14 was knocked down by RNAi in Sertoli cells in vitro and also in testicular cells in vivo have illustrated its role in conferring the integrity of actin filament bundles at the ES, perturbing the Sertoli cell tight junction (TJ)-pemeability barrier function in vitro, and also spermatid polarity and adhesion in vivo, thereby regulating spermatid transport at spermiation. Herein, we critically evaluate these earlier findings and also provide a likely hypothetic model based on the functional role of RAI14 at the ES, and how RAI14 is working with palladin and other actin regulatory proteins in the testis to regulate the transport of (1) spermatids and (2) preleptotene spermatocytes across the seminiferous epithelium and the blood-testis barrier (BTB), respectively, during spermatogenesis. This model should serve as a framework upon which functional experiments can be designed to better understand the biology of RAI14 and other actin-binding and regulatory proteins in the testis.

Adjudin disrupts spermatogenesis by targeting drug transporters: Lesson from the breast cancer resistance protein (BCRP).

For non-hormonal male contraceptives that exert their effects in the testis locally instead of via the hypothalamic-pituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII‒VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be synthesized can improve their efficacy in downregulating BCRP and perhaps other drug efflux transporters in the testis to improve their efficacy to traverse the BTB by modifying their interacting domains.

Breast cancer resistance protein (Bcrp) and the testis--an unexpected turn of events.

Breast cancer resistance protein (Bcrp) is an ATP-dependent efflux drug transporter. It has a diverse spectrum of hydrophilic and hydrophobic substrates ranging from anticancer, antiviral and antihypertensive drugs, to organic anions, antibiotics, phytoestrogens (e.g., genistein, daidzein, coumestrol), xenoestrogens and steroids (e.g., dehydroepiandrosterone sulfate). Bcrp is an integral membrane protein in cancer and normal cells within multiple organs (e.g., brain, placenta, intestine and testis) that maintains cellular homeostasis by extruding drugs and harmful substances from the inside of cells. In the brain, Bcrp is a major component of the blood-brain barrier located on endothelial cells near tight junctions (TJs). However, Bcrp is absent at the Sertoli cell blood-testis barrier (BTB); instead, it is localized almost exclusively to the endothelial TJ in microvessels in the interstitium and the peritubular myoid cells in the tunica propria. Recent studies have shown that Bcrp is also expressed stage specifically and spatiotemporally by Sertoli and germ cells in the seminiferous epithelium of rat testes, limited only to a testis-specific cell adhesion ultrastructure known as the apical ectoplasmic specialisation (ES) in stage VI-early VIII tubules. These findings suggest that Bcrp is equipped by late spermatids and Sertoli cells to protect late-stage spermatids completing spermiogenesis. Furthermore, Bcrp was found to be associated with F (filamentous)-actin and several actin regulatory proteins at the apical ES and might be involved in the organisation of actin filaments at the apical ES in stage VII-VIII tubules. These findings will be carefully evaluated in this brief review.

Actin cross-linking protein palladin and spermatogenesis.

In the seminiferous epithelium of the mammalian testis, the most distinctive ultrastructure is the extensive bundles of actin filaments that lie near the Sertoli-spermatid interface and the Sertoli-Sertoli cell interface known as the apical ectoplasmic specialization (apical ES) and the basal ES, respectively. These actin filament bundles not only confer strong adhesion at these sites, they are uniquely found in the testis. Recent studies have shown that ES also confers spermatid and Sertoli cell polarity in the seminiferous epithelium during the epithelial cycle. While these junctions were first described in the 1970s, there are few functional studies in the literature to examine the regulation of these actin filament bundles. It is conceivable that these actin filament bundles at the ES undergo extensive re-organization to accommodate changes in location of developing spermatids during spermiogenesis as spermatids are transported across the seminiferous epithelium. Additionally, these actin filaments are rapidly reorganized during BTB restructuring to accommodate the transit of preleptotene spermatocytes across the barrier at stage VIII of the epithelial cycle. Thus, actin binding and regulatory proteins are likely involved in these events to confer changes in F-actin organization at these sites. Interestingly, there are no reports in the field to study these regulatory proteins until recently. Herein, we summarize some of the latest findings in the field regarding a novel actin cross-linker and actin-bundling protein called palladin. We also discuss in this opinion article the likely role of palladin in regulating actin filament bundles at the ES during spermatogenesis, highlighting the significant of palladin and how this protein is plausibly working in concert with other actin-binding/regulatory proteins and components of polarity proteins to regulate the cyclic events of actin organization and re-organization during the epithelial cycle of spermatogenesis. We also propose a hypothetic model by which palladin regulates ES restructuring during the epithelial cycle of spermatogenesis.

Role of P-glycoprotein at the blood-testis barrier on adjudin distribution in the testis: a revisit of recent data.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in mammals including rodents and humans. It is used to sequester meiosis I and II, postmeiotic spermatid development via spermiogenesis and the release of sperm at spermiation from the systemic circulation, such that these events take place in an immune-privileged site in the adluminal (apical) compartment behind the BTB, segregated from the host immune system. Additionally, drug transporters, namely efflux (e.g., P-glycoprotein) and influx (e.g., Oatp3) pumps, many of which are integral membrane proteins in Sertoli cells at the BTB also work cooperatively to restrict the entry of drugs, toxicants, chemicals, steroids and other xenobiotics into the adluminal compartment. As such, the BTB that serves as an important physiological and selective barrier to protect germ cell development also poses a "hurdle" in male contraceptive development. For instance, adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, a potential nonhormonal male contraceptive that exerts its effects on germ cell adhesion, most notably at the Sertoli cell-spermatid interface, to induce "premature" germ cell loss from the seminiferous epithelium mimicking spermiation, has a relatively poor bioavailability largely because of the BTB. Since male contraceptives (e.g., adjudin) will be used by healthy men for an extended period of his life span after puberty, a better understanding on the BTB is necessary in order to effectively deliver drugs across this blood-tissue barrier in particular if these compounds exert their effects on developing germ cells in the adluminal compartment. This can also reduce long-term toxicity and health risk if the effective dosing can be lowered in order to widen the margin between its safety and efficacy. Herein, we summarize latest findings in this area of research, we also provide a critical evaluation on research areas that deserve attention in future studies.

Interactions of laminin ß3 fragment with ß1-integrin receptor: A revisit of the apical ectoplasmic specialization-blood-testis-barrier-hemidesmosome functional axis in the testis.

Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes across the immunological barrier near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin ß3 or γ3 chains) derived from the α6ß1-integrin-laminin333 protein complex at the apical ES, which were likely generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on ß1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and ß1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis.

Cancer/testis (CT) antigens, carcinogenesis and spermatogenesis.

During spermatogenesis, spermatogonial stem cells, undifferentiated and differentiated spermatogonia, spermatocytes, spermatids and spermatozoa all express specific antigens, yet the functions of many of these antigens remain unexplored. Studies in the past three decades have shown that many of these transiently expressed genes in developing germ cells are proto-oncogenes and oncogenes, which are expressed only in the testis and various types of cancers in humans and rodents. As such, these antigens are designated cancer/testis antigens (CT antigens). Since the early 1980s, about 70 families of CT antigens have been identified with over 140 members are known to date. Due to their restricted expression in the testis and in various tumors in humans, they have been used as the target of immunotherapy. Multiple clinical trials at different phases are now being conducted with some promising results. Interestingly, in a significant number of cancer patients, antibodies against some of these CT antigens were detected in their sera. However, antibodies against these CT antigens in humans under normal physiological conditions have yet to be reported even though many of these antigens are residing outside of the blood-testis barrier (BTB), such as in the basal compartment of the seminiferous epithelium and in the stem cell niche in the testis. In this review, we summarize latest findings in the field regarding several selected CT antigens which may be intimately related to spermatogenesis due to their unusual restricted expression during different discrete events of spermatogenesis, such as cell cycle progression, meiosis and spermiogenesis. This information should be helpful to investigators in the field to study the roles of these oncogenes in spermatogenesis.

Alpha2-macroglobulin expression in the liver in response to inflammation is mediated by the testis.

Earlier studies have shown that germ cells or germ cell-conditioned media are capable of regulating alpha2-macroglobulin (alpha2-MG, a non-specific protease inhibitor) expression by Sertoli cells and hepatocytes cultured in vitro. These results illustrate a possible physiological link between testes and liver regarding alpha2-MG production. Using a series of surgical procedures including castration, hemicastration, and hepatectomy coupled with Northern blot and immunoblot analyses, we report herein that the surge in alpha2-MG expression in the liver in response to inflammation is indeed regulated, at least in part, by the testis via testosterone. It was found that hepatectomy induced at least a tenfold increase in the steady-state mRNA and protein production of alpha2-MG in the liver. However, castration induced a mild but not statistically significant induction of alpha2-MG in the liver in contrast to sham operation or hemicastration alone, when hemicastration alone could induce liver alpha2-MG production by almost fourfold. Perhaps most important of all, hepatectomy accompanied by castration significantly reduced the liver alpha2-MG response to the surgery-induced inflammation compared with hepatectomy alone, illustrating that the removal of the testicles can induce a loss of signal communications between the testis and the liver, rendering a significant loss of the alpha2-MG response to experimentally induced inflammation in the liver. Interestingly, this lack of response of the liver to surgery-induced inflammation regarding alpha2-MG production following castration could be restored, at least in part, by using testosterone implants placed subdermally 6 days prior to orchiectomy. Collectively, these results illustrate that a physiological link does indeed exist between the testis and the liver, and that testes per se can influence the liver in vivo alpha2-MG expression in response to inflammation possibly via testosterone or testosterone-induced biological factor(s).