The human CIB1-EVER1-EVER2 complex governs keratinocyte-intrinsic immunity to ß-papillomaviruses.

Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human ß-papillomaviruses (ß-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to ß-HPVs of EV patients.

Expression of Programmed Cell Death Ligand in Cutaneous Squamous Cell Carcinoma and Treatment of Locally Advanced Disease With Pembrolizumab.

IMPORTANCE: Limited therapies are available in patients with inoperable locally advanced cutaneous squamous cell carcinoma (cSCC). OBJECTIVE: To determine the efficacy of programmed cell death 1 receptor (PD-1) inhibitors in locally advanced cSCC. DESIGN, SETTING, AND PARTICIPANTS: A single patient with locally advanced cSCC who declined surgery and radiotherapy underwent treatment with pembrolizumab, an anti-PD-1 antibody, at an academic dermatologic surgery section and cancer center. The patient was followed up for clinical and radiologic regression of cSCC. With the use of NanoString to amplify potential biomarkers, immunohistochemistry, and immunofluorescence, the ex vivo expression of PD-1 and a ligand (PD-L2) was assessed in 38 cSCC biopsy specimens from 24 patients with cSCC. Expression of PD-L1 and PD-L2 in the cSCC microenvironment was defined. INTERVENTION: Pembrolizumab, 2 mg/kg every 3 weeks, for 4 cycles. MAIN OUTCOMES AND MEASURES: Expression of PD-L1 and PD-L2 in the cSCC microenvironment. RESULTS: In 1 patient with locally advanced cSCC who was treated with pembrolizumab, nearly complete tumor regression was observed after 4 cycles of therapy. The NanoString technology used in 38 cSCC biopsy specimens from 24 patients with cSCC (19 men and 5 women; mean [SD] age, 76.4 [12.2] years) detected increased PD-1 and PD-L2 expression in high-risk cSCC. Immunohistochemical analysis confirmed enhanced expression of PD-1 and its ligands in cSCC with perineural invasion (mean [SEM] expression, 5.06 [1.27]; P = .05), superficial cSCC (mean [SEM] expression, 3.58 [1.50]; P = .15), organ transplant-associated cSCC (mean [SEM] expression, 3.01 [0.54]; P = .005), and infiltrative cSCC (mean [SD] expression, 2.01 [0.30]; P = .006) compared with normal skin specimens. In double-label immunofluorescence staining, CD11c+, a marker of myeloid dendritic cells, colocalized with PD-L1 and PD-L2 in cSCC lesions. CONCLUSIONS AND RELEVANCE: The favorable treatment response combined with significant involvement of PD-1 and PD ligands in cSCC lesions suggests that PD-1 blockade may be a viable therapeutic option for locally advanced cSCC and provides rationale for further investigation in future clinical trials.

Aberrant connective tissue differentiation towards cartilage and bone underlies human keloids in African Americans.

Keloids are benign fibroproliferative tumors more frequently found among African Americans. Until now, keloid etiopathogenesis is not fully understood. To characterize keloids in African Americans, we performed transcriptional profiling of biopsies from large chronic keloids, adjacent non-lesional (NL) skin (n=3) and a newly formed keloid lesion using Affymetrix HGU133 2.0 plus arrays. Quantitative RT-PCR (qRT-PCR) and immunohistochemistry (IHC) staining were performed to confirm increased expression of relevant genes. We identified 1202 upregulated and 961 downregulated differentially expressed genes (DEGs) between keloid and NL skin; 1819 up- and 1867 downregulated DEGs between newly formed keloid and NL skin; and 492 up- and 775 downregulated DEGs between chronic and newly formed keloid (fold change >2, false discovery rate <0.05). Many of the top upregulated DEGs between chronic keloid and NL skin and between newly formed keloid and NL skin are involved in bone/cartilage formation including Fibrillin 2 (FBN2), Collagen type X alpha 1, Asporin (ASPN), Cadherin 11 (CDH11), Bone morphogenic protein 1 (BMP1), Secreted phosphoprotein 1 and Runt-related transcription factor 2 (RUNX2). qRT-PCR confirmed significant (P<.05) upregulation of BMP1, RUNX2, CDH11 and FBN2 in chronic keloid compared to NL skin. IHC staining showed increased protein expression of ASPN, CDH11, BMP1 and RUNX2 on chronic and newly formed keloid compared to NL skin. Our study shows that large keloids in African Americans represent a dysplasia of cutaneous connective tissue towards immature cartilage or bone differentiation. The phenotype is potentially regulated by overexpression of RUNX2. This knowledge may give insights to guide the development of better treatment for the disease in the future.

Discrimination of Dysplastic Nevi from Common Melanocytic Nevi by Cellular and Molecular Criteria.

Dysplastic nevi (DNs), also known as Clark's nevi or atypical moles, are distinguished from common melanocytic nevi by variegation in pigmentation and clinical appearance, as well as differences in tissue patterning. However, cellular and molecular differences between DNs and common melanocytic nevi are not completely understood. Using cDNA microarray, quantitative RT-PCR, and immunohistochemistry, we molecularly characterized DNs and analyzed the difference between DNs and common melanocytic nevi. A total of 111 probesets (91 annotated genes, fold change > 2.0 and false discovery rate < 0.25) were differentially expressed between the two lesions. An unexpected finding in DNs was altered differentiation and activation of epidermal keratinocytes with increased expression of hair follicle-related molecules (keratin 25, trichohyalin, ribonuclease, RNase A family, 7) and inflammation-related molecules (S100A7, S100A8) at both genomic and protein levels. The immune microenvironment of DNs was characterized by an increase of T helper type 1 (IFNγ) and T helper type 2 (IL13) cytokines as well as an upregulation of oncostatin M and CXCL1. DUSP3, which regulates cellular senescence, was identified as one of the disease discriminative genes between DNs and common melanocytic nevi by three independent statistical approaches and its altered expression was confirmed by immunohistochemistry. The molecular and cellular changes in which the epidermal-melanin unit undergoes follicular differentiation as well as upregulation of defined cytokines could drive complex immune, epidermal, and pigmentary alterations.

Deficiency of CDKN1A or both CDKN1A and CDKN1B affects the pubertal development of mouse Leydig cells.

Cyclin-dependent kinase inhibitors p21(Cip1) (CDKN1A) and p27(Kip1) (CDKN1B) are expressed in Leydig cells. Previously, we reported that Cdkn1b knockout in the mouse led to increased Leydig cell proliferative capacity and lower steroidogenesis. However, the relative importance of CDKN1A and CDKN1B in these regulations was unclear. In the present study, we examined the relative importance of CDKN1A and CDKN1B in regulation of Leydig cell proliferation and steroidogenesis by whole-body knockout of CDKN1A (Cdkn1a(-/-)) and CDKN1A/CDKN1B double knockout (DBKO). The cell number, 5-bromo-2-deoxyuridine incorporation rate, steroidogenesis, and steroidogenic enzyme mRNA levels and activities of Leydig cells were compared among wild-type (WT), Cdkn1a(-/-), and DBKO mice. Relative to WT mice, Leydig cell number per testis was doubled in the DBKO and unchanged in the Cdkn1a(-/-) mice. Testicular testosterone levels and mRNA levels for luteinizing hormone receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 17alpha-hydroxylase/17,20-lyase (Cyp17a1), and 17beta-hydroxysteroid dehydrogenase 3 (Hsd17b3) and their respective proteins were significantly lower in the DBKO mice. However, testicular testosterone level was unchanged in the Cdkn1a(-/-) mice, although Lhcgr mRNA levels were significantly lower relative to those in the WT control. We conclude that Cdkn1a(-/-) did not increase Leydig cell numbers (although a defect of Leydig cell function was noted), whereas DBKO caused a significant increase of Leydig cell numbers but a decrease of steroidogenesis.

Identification of anaplastic lymphoma kinase as a potential therapeutic target in Basal Cell Carcinoma.

The pathogenesis of BCC is associated with sonic hedgehog (SHH) signaling. Vismodegib, a smoothened inhibitor that targets this pathway, is now in clinical use for advanced BCC patients, but its efficacy is limited. Therefore, new therapeutic options for this cancer are required. We studied gene expression profiling of BCC tumour tissues coupled with laser capture microdissection to identify tumour specific receptor tyrosine kinase expression that can be targeted by small molecule inhibitors. We found a >250 fold increase (FDR<10-4) of the oncogene, anaplastic lymphoma kinase (ALK) as well as its ligands, pleiotrophin and midkine in BCC compared to microdissected normal epidermis. qRT-PCR confirmed increased expression of ALK (p<0.05). Stronger expression of phosphorylated ALK in BCC tumour nests than normal skin was observed by immunohistochemistry. Crizotinib, an FDA-approved ALK inhibitor, reduced keratinocyte proliferation in culture, whereas a c-Met inhibitor did not. Crizotinib significantly reduced the expression of GLI1 and CCND2 (members of SHH-pathway) mRNA by approximately 60% and 20%, respectively (p<0.01). Our data suggest that ALK may increase GLI1 expression in parallel with the conventional SHH-pathway and promote keratinocyte proliferation. Hence, an ALK inhibitor alone or in combination with targeting SHH-pathway molecules may be a potential treatment for BCC patients.

Effects of luteinizing hormone and androgen on the development of rat progenitor Leydig cells in vitro and in vivo.

Progenitor Leydig cells are derived from stem cells. The proliferation and differentiation of progenitor Leydig cells significantly contributes to Leydig cell number during puberty. However, the regulation of these processes remains unclear. The objective of the present study was to determine whether luteinizing hormone (LH) or androgen contributes to the proliferation and differentiation of progenitor Leydig cells. Fourteen-day-old male Sprague-Dawley rats were treated for 7 days with NalGlu, which is a gonadotropin-releasing hormone antagonist, to reduce the secretion of LH in the pituitary and thus, androgen in the testis. Rats were co-administered with LH or 7α-methyl-nortestosterone (MENT), which is an androgen resistant to metabolism by 5α-reductase 1 in progenitor Leydig cells, and the subsequent effects of LH or androgen were measured. (3)H-Thymidine was also intravenously injected into rats to study thymidine incorporation in progenitor Leydig cells. Progenitor Leydig cells were examined. NalGlu administration reduced progenitor Leydig cell proliferation by 83%. In addition, LH or MENT treatment restored Leydig cell proliferative capacity to 73% or 50% of control, respectively. The messenger RNA levels of proliferation-related genes were measured using real-time PCR. The expression levels of Igf1, Lifr, Pdgfra, Bcl2, Ccnd3 and Pcna were upregulated by MENT, and those of Pdgfra, Ccnd3 and Pcna were upregulated by LH. Both LH and MENT stimulated the differentiation of progenitor Leydig cells in vitro. We concluded that both LH and MENT were involved in regulating the development of progenitor Leydig cells.

IL-17 and TNF synergistically modulate cytokine expression while suppressing melanogenesis: potential relevance to psoriasis.

Inflammation-associated pigmentation changes are extremely common, but the etiology underlying this clinical observation remains elusive. Particularly, it is unclear how the myriad of cytokines known to be involved in inflammatory skin processes affect epidermal melanocytes. We sought to determine how IL-17 and tumor necrosis factor (TNF) influence normal human melanocytes, as these two cytokines have been implicated in various skin diseases. IL-17 and TNF jointly stimulated broad inductions of cytokines, including melanoma mitogens CXCL1 and IL-8. Moreover, IL-17 and TNF synergistically inhibited pigmentation-related signaling and melanin production, and induced keratinocyte production of ß-defensin 3, an antagonist for melanocortin 1 receptor. When analyzing psoriasis lesions that are known to overexpress IL-17 and TNF, we observed an increase in melanocyte number and a simultaneous decrease in pigmentation signaling. Furthermore, therapeutic neutralization of TNF and IL-17 with mAbs resulted in a rapid recovery of pigment gene expression in psoriasis lesions. These results demonstrate that IL-17 and TNF can affect both the growth and pigment production of melanocytes, which may contribute to the pigmentation changes associated with psoriasis. These findings may allow the development of novel therapeutics for pigmentary disorders and bring new insights into the immune milieu surrounding melanocytes and related neoplasms.

Increased Tc22 and Treg/CD8 ratio contribute to aggressive growth of transplant associated squamous cell carcinoma.

Immune suppressed organ transplant recipients suffer increased morbidity and mortality from primary cutaneous SCC. We studied tumor microenvironment in transplant-associated SCC (TSCC), immune-competent SCC and normal skin by IHC, IF and RT-PCR on surgical discard. We determined T cell polarization in TSCC and SCC by intracellular cytokine staining of T cell crawl outs from human skin explants. We studied the effects of IL-22, an inducer of keratinocyte proliferation, on SCC proliferation in vitro. SCC and TSCC are both associated with significantly higher numbers of CD3(+) and CD8(+) T cells compared to normal skin. TSCC showed a higher proportion of Foxp3(+) T regs to CD8(+) T cells compared to SCC and a lower percentage of IFN-γ producing CD4(+) T cells. TSCC, however, had a higher percentage of IL-22 producing CD8(+) T cells compared to SCC. TSCC showed more diffuse Ki67 and IL-22 receptor (IL-22R) expression by IHC. IL-22 induced SCC proliferation in vitro despite serum starvation. Diminished cytotoxic T cell function in TSCC due to decreased CD8/T-reg ratio may permit tumor progression. Increased IL-22 and IL-22R expression could accelerate tumor growth in transplant patients. IL-22 may be an attractive candidate for targeted therapy of SCC without endangering allograft survival.

Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment.

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17ß, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17ß-hydroxysteroid dehydrogenase 3 and 11ß-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17ß-hydroxysteroid dehydrogenase 3 and 11ß-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

CD200 upregulation in vascular endothelium surrounding cutaneous squamous cell carcinoma.

OBJECTIVE: To characterize the presence of CD200 and CD200 receptor (CD200R) in the human cutaneous squamous cell carcinoma (SCC) microenvironment and to define a possible role for the CD200 axis in immune evasion by SCC. DESIGN: Gene expression in SCC vs normal skin was studied. Laser capture microdissection was used to determine differential expression of CD200 in normal skin vs actinic keratosis vs SCC in situ vs invasive SCC. Immunofluorescence microscopy was used to define expression of CD200R on macrophages, myeloid dendritic cells, natural killer cells, and T cells in SCC vs normal skin. The effects of SCC supernatant on induction of CD200 in human blood endothelial cells was also examined. SETTING: Academic Medical Center with an established Section of Mohs and Dermatologic Surgery and an active Cutaneous Biology Research Program. PARTICIPANTS: Surgical discard tissue from deidentified patients and samples of normal skin from healthy volunteers were used in this study. MAIN OUTCOME MEASURES: Expression of CD200 on SCC-associated blood vessels; expression of CD200 receptor on SCC-associated macrophages and T cells; and induction of CD200 on endothelial cells by SCC supernatants. RESULTS: CD200 gene and message were upregulated in SCC stroma. Immunostaining revealed a higher number of CD200(+) cells in SCC stroma than in normal dermis (180.8 cells/mm(2) vs 24.6 cells/mm(2)) (P<.01). CD200 was further identified mainly on blood vessel endothelium in SCC. Tumor supernatant was able to induce CD200 expression on human dermal blood endothelial cells in culture. CD200R was identified on macrophages and dendritic cells in SCC microenvironment. CONCLUSIONS: CD200 expression on local blood vessels may promote tumor progression by suppressing CD200R myeloid cells during diapedesis. These data highlight a previously unrecognized mechanism of immune evasion by SCC and may provide guidance for the development of targeted therapy.

Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common disease with an increasing prevalence. The primary pathogenesis of the disease is still elusive, resulting in the lack of specific treatments. AD is currently considered a biphasic disease, with T(H)2 predominating in acute disease and a switch to T(H)1 characterizing chronic disease. Elucidation of the molecular factors that participate in the onset of new lesions and maintenance of chronic disease is critical for the development of targeted therapeutics. OBJECTIVES: We sought to characterize the mechanisms underlying the onset and maintenance of AD. METHODS: We investigated intrapersonal sets of transcriptomes from nonlesional skin and acute and chronic lesions of 10 patients with AD through genomic, molecular, and cellular profiling. RESULTS: Our study associated the onset of acute lesions with a striking increase in a subset of terminal differentiation proteins, specifically the cytokine-modulated S100A7, S100A8, and S100A9. Acute disease was also associated with significant increases in gene expression levels of major T(H)22 and T(H)2 cytokines and smaller increases in IL-17 levels. A lesser induction of T(H)1-associated genes was detected in acute disease, although some were significantly upregulated in chronic disease. Further significant intensification of major T(H)22 and T(H)2 cytokines was observed between acute and chronic lesions. CONCLUSIONS: Our data identified increased S100A7, S100A8, and S100A9 gene expression with AD initiation and concomitant activation of T(H)2 and T(H)22 cytokines. Our findings support a model of progressive activation of T(H)2 and T(H)22 immune axes from the acute to chronic phases, expanding the prevailing view of pathogenesis with important therapeutic implications.

Th17 cells and activated dendritic cells are increased in vitiligo lesions.

BACKGROUND: Vitiligo is a common skin disorder, characterized by progressive skin de-pigmentation due to the loss of cutaneous melanocytes. The exact cause of melanocyte loss remains unclear, but a large number of observations have pointed to the important role of cellular immunity in vitiligo pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized T cell and inflammation-related dermal dendritic cell (DC) subsets in pigmented non-lesional, leading edge and depigmented lesional vitiligo skin. By immunohistochemistry staining, we observed enhanced populations of CD11c+ myeloid dermal DCs and CD207+ Langerhans cells in leading edge vitiligo biopsies. DC-LAMP+ and CD1c+ sub-populations of dermal DCs expanded significantly in leading edge and lesional vitiligo skin. We also detected elevated tissue mRNA levels of IL-17A in leading edge skin biopsies of vitiligo patients, as well as IL-17A positive T cells by immunohistochemistry and immunofluorescence. Langerhans cells with activated inflammasomes were also noted in lesional vitiligo skin, along with increased IL-1ß mRNA, which suggest the potential of Langerhans cells to drive Th17 activation in vitiligo. CONCLUSIONS/SIGNIFICANCE: These studies provided direct tissue evidence that implicates active Th17 cells in vitiligo skin lesions. We characterized new cellular immune elements, in the active margins of vitiligo lesions (e.g. populations of epidermal and dermal dendritic cells subsets), which could potentially drive the inflammatory responses.

Tumor-associated macrophages in the cutaneous SCC microenvironment are heterogeneously activated.

Tumor-associated macrophages (TAMs) may have an important role in tumor immunity. We studied the activation state of TAMs in cutaneous SCC, the second most common human cancer. CD163 was identified as a more abundant, sensitive, and accurate marker of TAMs when compared with CD68. CD163(+) TAMs produced protumoral factors, matrix metalloproteinases 9 and 11 (MMP9 and MMP11), at the gene and protein levels. Gene set enrichment analysis (GSEA) was used to evaluate M1 and M2 macrophage gene sets in the SCC genes and to identify candidate genes in order to phenotypically characterize TAMs. There was coexpression of CD163 and alternatively activated "M2" markers, CD209 and CCL18 (chemokine (C-C motif) ligand 18). There was enrichment for classically activated "M1" genes in SCC, which was confirmed in situ by colocalization of CD163 and phosphorylated STAT1 (signal transducer and activator of transcription 1), IL-23p19, IL-12/IL-23p40, and CD127. Also, a subset of TAMs in SCC was bi-activated as CD163(+) cells expressed markers for both M1 and M2, shown by triple-label immunofluorescence. These data support heterogeneous activation states of TAMs in SCC, and suggest that a dynamic model of macrophage activation would be more useful to characterize TAMs.

A seamless trespass: germ cell migration across the seminiferous epithelium during spermatogenesis.

During spermatogenesis, preleptotene spermatocytes traverse the blood-testis barrier (BTB) in the seminiferous epithelium, which is reminiscent of viral pathogens breaking through the tight junctions of host epithelial cells. The process also closely resembles the migration of leukocytes across endothelial tight junctions to reach inflammation sites. Cell adhesion molecules of the immunoglobulin superfamily (e.g., JAM/CAR/nectin) participate in germ cell migration by conferring transient adhesion between Sertoli and germ cells through homophilic and heterophilic interactions. The same molecules also comprise the junctional complexes at the BTB. Interestingly, JAM/CAR/nectin molecules mediate virus uptake and leukocyte transmigration in strikingly similar manners. It is likely that the strategy used by viruses and leukocytes to break through junctional barriers is used by germ cells to open up the inter-Sertoli cell junctions. In associating these diverse cellular events, we highlight the "guiding" role of JAM/CAR/nectin molecules for germ cell passage. Knowledge on viral invasion and leukocyte transmigration has also shed insights into germ cell movement during spermatogenesis.

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface.

The coxsackie and adenovirus receptor (CAR), a putative cell-cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood-testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFalpha treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli-germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.

Dynamin II interacts with the cadherin- and occludin-based protein complexes at the blood-testis barrier in adult rat testes.

In adult rat testes, blood-testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient 'opening'. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), beta-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/beta-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with beta-catenin and ZO-1. Beta-catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an 'engaged'state to reinforce the barrier function in normal testes. However, beta-catenin and ZO-1 were 'disengaged' from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the 'disengagement' of beta-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.

Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes.

The timely restructuring of the blood-testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor alpha (TNFalpha), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFalpha directly to adult rat testes in vivo at 0.5 and 2 mug/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, alpha-, and beta-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFalpha treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFalpha by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring ('opening'?) of the BTB during spermatogenesis to facilitate germ cell migration.