Isolation and Characterization of Neutralizing Monoclonal Antibodies from a Large Panel of Murine Antibodies against RBD of the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

COVID-eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

The Nuts and Bolts of SARS-CoV-2 Spike Receptor-Binding Domain Heterologous Expression.

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

Structural basis of ubiquitination mediated by protein splicing in early Eukarya.

BACKGROUND: Inteins are intervening proteins, which are known to perform protein splicing. The reaction results in the production of an intein domain and an inteinless protein, which shows no trace of the insertion. BIL2 is part of the polyubiquitin locus of Tetrahymena thermophila (BUBL), where two bacterial-intein-like (BIL) domains lacking the C + 1 nucleophile, are flanked by two independent ubiquitin-like domains (ubl4/ubl5). METHODS: We solved the X-ray structures of BIL2 in both the inactive and unprecedented, zinc-induced active, forms. Then, we characterized by mass spectrometry the BUBL splicing products in the absence and in the presence of T.thRas-GTPase. Finally, we investigated the effect of ubiquitination on T.thRas-GTPase by molecular dynamics simulations. RESULTS: The structural analysis demonstrated that zinc-induced conformational change activates protein splicing. Moreover, mass spectrometry characterization of the splicing products shed light on the possible function of BIL2, which operates as a "single-ubiquitin-dispensing-platform", allowing the conjugation, via isopeptide bond formation (K(εNH2)-C-ter), of ubl4 to either ubl5 or T.thRas-GTPase. Lastly, we demonstrated that T.thRas-GTPase ubiquitination occurs in proximity of the nucleotide binding pocket and stabilizes the protein active state. CONCLUSIONS: We demonstrated that BIL2 is activated by zinc and that protein splicing induced by this intein does not take place through classical or aminolysis mechanisms but via formation of a covalent isopeptide bond, causing the ubiquitination of endogenous substrates such as T.thRas-GTPase. GENERAL SIGNIFICANCE: In this "enzyme-free" ubiquitination mechanism the isopeptide formation, which canonically requires E1-E2-E3 enzymatic cascade and constitutes the alphabet of ubiquitin biology, is achieved in a single, concerted step without energy consumption.

Roles of Sorcin in Drug Resistance in Cancer: One Protein, Many Mechanisms, for a Novel Potential Anticancer Drug Target.

The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named "resistance-related" because of its co-amplification with ABCB1. Although for years sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that sorcin plays an important part in MDR, indicating a possible role of sorcin as an oncoprotein. The present review illustrates sorcin roles in the generation of MDR via many mechanisms and points to sorcin as a novel potential target of different anticancer molecules.

NMR structure of a non-conjugatable, ADP-ribosylation associated, ubiquitin-like domain from Tetrahymena thermophila polyubiquitin locus.

BACKGROUND: Ubiquitin-like domains (UbLs), in addition to being post-translationally conjugated to the target through the E1-E2-E3 enzymatic cascade, can be translated as a part of the protein they ought to regulate. As integral UbLs coexist with the rest of the protein, their structural properties can differ from canonical ubiquitin, depending on the protein context and how they interact with it. In this work, we investigate T.th-ubl5, a UbL present in a polyubiquitin locus of Tetrahymena thermophila, which is integral to an ADP-ribosyl transferase protein. Only one other co-occurrence of these two domains within the same protein has been reported. METHODS: NMR, multiple sequence alignment, MD simulations and SPR have been used to characterize the structure of T.th-ubl5, identify putative binders and experimentally test the interaction, respectively. RESULTS: Molecular dynamics simulations showed that T.th-ubl5 is unable to bind the proteasome like ubiquitin due to the lack of the conserved hydrophobic patch. Of other integral UbLs identified by structural and sequence alignment, T.th-ubl5 showed high structural and sequence resemblance with the Ras-binding epitope of FERM UbLs. SPR experiments confirmed that a strong and specific interaction occurs between T.th-ubl5 and T.th-Ras. CONCLUSION: Data indicate that T.th-ubl5 does not interact with the proteasome like ubiquitin but acts as a decoy for the recruitment of Ras protein by the ADP-ribosyl transferase domain. GENERAL SIGNIFICANCE: Mono-ADP-ribosylation of Ras proteins is known as a prerogative of bacterial toxins. T.th-ubl5 mediated recruitment of Ras highlights the possibility of an unprecedented post-translational modification with interesting implication for signalling pathways.

Sorcin, a calcium binding protein involved in the multidrug resistance mechanisms in cancer cells.

Sorcin is a penta-EF hand calcium binding protein, which participates in the regulation of calcium homeostasis in cells. Sorcin regulates calcium channels and exchangers located at the plasma membrane and at the endo/sarcoplasmic reticulum (ER/SR), and allows high levels of calcium in the ER to be maintained, preventing ER stress and possibly, the unfolded protein response. Sorcin is highly expressed in the heart and in the brain, and overexpressed in many cancer cells. Sorcin gene is in the same amplicon as other genes involved in the resistance to chemotherapeutics in cancer cells (multi-drug resistance, MDR) such as ABCB4 and ABCB1; its overexpression results in increased drug resistance to a number of chemotherapeutic agents, and inhibition of sorcin expression by sorcin-targeting RNA interference leads to reversal of drug resistance. Sorcin is increasingly considered a useful marker of MDR and may represent a therapeutic target for reversing tumor multidrug resistance.

Penile doppler ultrasound in patients with erectile dysfunction (ED): role of peak systolic velocity measured in the flaccid state in predicting arteriogenic ED and silent coronary artery disease.

INTRODUCTION: The use of the penile peak systolic velocity (PSV) measured in the flaccid state during penile color Doppler ultrasound (PCDU) examination has been questioned without substantial evidence. AIM: To assess the validity of PSV measured in the flaccid state during PCDU, in patients consulting for erectile dysfunction (ED). METHODS: A consecutive series of 1,346 (mean age 55.0 +/- 12.0 years) male patients was studied. MAIN OUTCOMES MEASURES: All patients underwent PCDU performed both in the flaccid state and dynamic (after prostaglandin E1 stimulation) conditions. A subset of 20 subjects with uncomplicated type 2 diabetes underwent diagnostic testing for silent coronary heart disease by means of adenosine stress myocardial perfusion scintigraphy (SPECT). In these subjects penile arterial flow was simultaneously assessed by PCDU before and after systemic adenosine administration. RESULTS: Flaccid PSV showed a significant (r = 0.513, P < 0.0001) correlation with dynamic PSV. Receiver operating characteristic (ROC) curve analysis demonstrated that when a threshold of 13 cm/seconds was chosen, flaccid PSV was predictive for dynamic PSV < 25 and <35 cm/seconds with an accuracy of 89% and 82%, respectively. Among the subset of patients who underwent SPECT, an impaired coronary flow reserve (ICFR) occurred in nine cases (45%). When the same threshold of <13 cm/seconds was chosen, PSV before SPECT was predictive of ICFR with an accuracy of 80% (area under the ROC curve = 0.798 +/- 0.10; P < 0.05). After adjustment for confounders, anxiety symptoms were related to dynamic PSV (Adj. r = -0.154, P < 0.05) but not to flaccid PSV. CONCLUSIONS: Our results show that flow in the cavernosal arteries can be routinely evaluated by PCDU in the flaccid state. Performing PCDU only in the flaccid state allows identifying subjects with pathological dynamic PSV with accuracy higher than 80%. Furthermore, our preliminary data suggest that the same examination could identify diabetic subjects with ICFR with an accuracy of 80%.

Papillary carcinoma of a thyroglossal duct cyst in a patient with thyroid hemiagenesis: effectiveness of conservative surgical treatment.

OBJECTIVE: To describe a case of thyroglossal duct cyst carcinoma that arose in a patient with right thyroid lobe hemiagenesis. METHODS: We present the imaging, physical examination findings, treatment, and clinical course of the study patient. RESULTS: A 35-year-old woman was evaluated for a neck mass that had been present for 6 months and was slowly growing. She reported a previous diagnosis of right hemithyroid agenesis. The patient's preoperative workup included ultrasonography of the neck and head and neck T1- and T2-weighted magnetic resonance imaging, which showed right hemithyroid agenesis and a cystic lesion in the median region of the neck below the hyoid bone. Findings from chest x-rays and thyroid function tests were normal. The patient underwent a modified Sistrunk procedure that included removal of the median portion of the hyoid bone. Histologic findings showed a 2.5-cm thyroglossal duct cyst with a 0.6-cm focus of follicular variant of papillary carcinoma with invasion of the cyst wall. Total thyroidectomy was not performed because of the absence of tumoral invasion of the parenchyma around the thyroglossal duct cyst and because the patient was at low risk for aggressive disease. Cervical ultrasonography examinations were performed 6, 12, and 24 months after treatment, and all findings were normal. Presently, the patient is symptom-free after 4 years of follow-up and has no evidence of disease. CONCLUSION: Incidentally discovered, well-differentiated thyroid cancer that is confined to a thyroglossal duct cyst in a patient at low risk for aggressive disease can be adequately treated by a modified Sistrunk procedure that includes the median portion of the hyoid bone.

Different testosterone levels are associated with ejaculatory dysfunction.

INTRODUCTION: The role of testosterone (T) in pathogenesis of ejaculatory symptoms has not been completely clarified. AIM: To evaluate the possible contribution of T and hypogonadism in the control of the ejaculatory reflex, comparing subjects with premature ejaculation (PE) or delayed ejaculation (DE) to those without ejaculatory dysfunction. METHODS: A consecutive series of 2,437 (mean age 51.9 +/- 13.0 years) male patients with sexual dysfunction was studied. MAIN OUTCOME MEASURE: Several hormonal and biochemical parameters were studied, along with the structured interview on erectile dysfunction (SIEDY) structured interview. Hypogonadism were defined when total testosterone (TT) was lower than 10.4 nmol/L. RESULTS: Among the patients studied, 714 (25.9%) and 121 (4.4%) reported PE and DE, respectively. In the youngest age band (25-40 years), subjects with PE reported higher TT and free testosterone (FT) levels when compared to the other groups (subjects with DE or those without PE and DE; P < 0.05 for both). Conversely, in the oldest age band (55-70 years), lower TT and FT levels were observed in DE subjects. Accordingly, patients with PE showed the lowest (12%) and subjects with DE the highest (26%) prevalence of hypogonadism. These differences were confirmed even after adjustment for confounders such as age and libido (HR = 0.75 [0.57-0.99] and 1.83 [1.14-3.94] for PE and DE, respectively; both P < 0.05). CONCLUSIONS: Our data seem to suggest that T plays a facilitatory role in the control of ejaculatory reflex. Both central and peripheral mechanisms have been advocated to explain this association. Clinical studies are currently in progress to further establish the role of T in the ejaculatory dysfunction, attempting to revert DE by androgen administration.

NCEP-ATPIII-defined metabolic syndrome, type 2 diabetes mellitus, and prevalence of hypogonadism in male patients with sexual dysfunction.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS) are characterized by insulin resistance and often associated with male hypogonadism. AIM: To discriminate the specific contribution of T2DM and MetS to male hypogonadism. METHODS: A consecutive series of 1,134 (mean age 52.1 +/- 13 years) male patients with sexual dysfunction was studied. MAIN OUTCOME MEASURES: Several hormonal and biochemical parameters were studied along with ANDROTEST, a 12-item validated structured interview, specifically designed for the screening of hypogonadism (total testosterone [TT] <10.4 nmol/L or free testosterone [FT] <37 pmol/L) in a male population with sexual dysfunction. RESULTS: Irrespective of the criteria used to define hypogonadism, MetS was associated with a significantly higher prevalence of the condition, both in subjects with and without T2DM (41% and 29% vs. 13.2% and 77.1% and 58% vs. 40.6%, respectively, for TT and FT in patients with MetS and with or without T2DM, when compared with subjects without MetS and T2DM; both P < 0.0001). Conversely, T2DM was associated with a higher prevalence of hypogonadism in subjects with MetS but not in those without MetS. Patients with MetS, with or without T2DM, also showed a higher ANDROTEST score when compared with patients without MetS. Logistic multivariate regression analysis, incorporating the five components of MetS, identified a significant association of elevated waist circumference and hypertriglyceridemia with hypogonadism both in patients, with or without T2DM. CONCLUSIONS: Our study demonstrated that MetS, and in particular visceral adiposity (as assessed by increased waistline and hypertriglyceridemia), is specifically associated with hypogonadism in subjects consulting for sexual dysfunction.

A comparison of NCEP-ATPIII and IDF metabolic syndrome definitions with relation to metabolic syndrome-associated sexual dysfunction.

INTRODUCTION: Metabolic syndrome (MetS) is a clustering of cardiovascular and metabolic risk factors, often associated with erectile dysfunction (ED) and hypogonadism. Recently, the International Diabetes Federation (IDF) proposed a substantial revision of the National Cholesterol Education Program-Third Adult Treatment Panel (NCEP-ATPIII) MetS criteria, essentially lowering the diagnostic cutoff values. Aim. To investigate the associations between these two recently proposed definitions of MetS with the relative risk of arteriogenic ED and hypogonadism in a large cohort of patients with male sexual dysfunction. METHODS: A consecutive series of 1086 patients with sexual dysfunction (mean age 51.9 +/- 12.8 years) was studied. MAIN OUTCOME MEASURES: Several hormonal, biochemical, and instrumental (penile Doppler ultrasound) parameters were studied, along with ANDROTEST, a 12-item validated structured interview, specifically designed for the screening hypogonadism in a sexual dysfunction population. In particular, a score >8 is predictive of low testosterone (<10.4 nmol/L) with a sensitivity and specificity of about 70%. RESULTS: The prevalence of MetS was 32.0% and 44.7% according to NCEP-ATPIII and IDF criteria, respectively. After adjustment for confounding factors, only NCEP-ATPIII was significantly associated with dynamic prostaglandin E(1)-stimulated penile flow (Vpmax, B = -7.7 +/- 3.8; P < 0.05). Patients with MetS defined according to both criteria reported lower total and free testosterone levels, higher prevalence of hypogonadism, and higher ANDROTEST score. However, when IDF, but not NCEP-ATPIII, criteria were fulfilled, the prevalence of hypogonadism was significantly lower than that observed in patients fulfilling both criteria (15.6% vs. 34.8%, respectively; P < 0.00001). Conversely, patients fulfilling NCEP-ATPIII, but not IDF, criteria did not show a significant different prevalence of hypogonadism than those positive for both sets of criteria (30.8% vs. 34.8%; P = NS). CONCLUSIONS: In patients with ED, NCEP-ATPIII criteria seem to be a better predictor of hypogonadism and impaired penile blood flow than IDF ones.

Assessment of the relational factor in male patients consulting for sexual dysfunction: the concept of couple sexual dysfunction.

In a consecutive series of 1140 male subjects reporting a stable couple relationship and different degrees of sexual dysfunction, we evaluated the impact of relational factors, as assessed by the Structured Interview on Erectile Dysfunction (SIEDY) Scale 2 (exploring, as reported by the patient, menopausal symptoms, partner's medical illness interfering with sexual activity, and reduced partner desire and climax). We found that the SIEDY Scale 2 is significantly and independently associated with erectile dysfunction (ED), delayed ejaculation, hypoactive sexual desire, and decreased frequency of intercourse. SIEDY Scale 2 scores are associated with an advanced age of the partner and a long couple relationship, independently from patient's age. In addition, an increased relational factor correlates with increased extramarital affairs, conflicts in the couple, alcohol abuse, and presence of depressive symptoms, as assessed by the Middlesex Hospital Questionnaire. Our results should encourage the andrologist to consider the context in which sexual symptoms develop, analyzing the relationship and partner's behaviors and diseases. Resolving, or at least ameliorating, the relational background and the sexual framework might help in treating male sexual dysfunction.

ANDROTEST: a structured interview for the screening of hypogonadism in patients with sexual dysfunction.

INTRODUCTION: Detecting hypogonadism, which is important in the general population, becomes crucial in patients with sexual dysfunctions, because hypogonadism can have a causal role for them and testosterone (T) substitution represents a milestone for the therapy. AIM: No inventories are available for the screening of hypogonadism in patients with sexual dysfunction. We wished to set up a brief structured interview providing scores useful for detecting hypogonadism defined as low total T (<10.4 nmol/L, 300 ng/dL) in a symptomatic population (sexual dysfunction). METHODS: A minimum set of items was identified within a larger structured interview through iterative receiver-operating characteristic curve analysis, with assessment of sensitivity and specificity for hypogonadism in a sample of 215 patients. MAIN OUTCOME MEASURES: Sensitivity and specificity were verified in a further sample of 664 patients. Correlation of test scores with prostate-specific antigen (PSA), testis volume, and others clinical and psychological parameters, was assessed for concurrent validity. RESULTS: In the validation sample, the final 12-item version of the interview (ANDROTEST) had a sensitivity and specificity of 68% and 65%, in detecting low total T (<10.4 nmol/L) and of 71% and 65%, in the screening for low free T (<37 pmol/L). Furthermore, patients with a pathological test (i.e., score >8) showed higher prevalence of hypogonadism-related signs, such as lower testis volume and higher depressive symptoms. Finally, when only younger patients (<54 years, which represents the median age of the sample) were considered, Log10 [PSA] levels were significantly lower in those with ANDROTEST score >8. CONCLUSION: ANDROTEST is a quick and easy-to-administer interview that provides scores for the screening of male hypogonadism in patients with sexual dysfunction.

Psychobiologic correlates of the metabolic syndrome and associated sexual dysfunction.

OBJECTIVES: The association of low testosterone level and erectile dysfunction (ED) with metabolic syndrome (MS) is receiving increasing attention. The present study determined the psychobiologic characteristics of sexual dysfunction (SD) associated with MS (as defined by the National Cholesterol Education Program's Adult Treatment Panel III criteria) in a series of 803 consecutive male outpatients. METHODS: Several hormonal, biochemical, and instrumental (penile Doppler ultrasound [PDU]) parameters were studied, along with general psychopathology scores (Middlesex Hospital Questionnaire modified [MHQ]). The Structured Interview on Erectile Dysfunction (SIEDY) was also applied. RESULTS: Among the 236 patients (29.4%) diagnosed as having a MS, 96.5% reported ED, 39.6% hypoactive sexual desire (HSD), 22.7% premature ejaculation, and 4.8% delayed ejaculation. Patients with MS were characterised by greater subjective (as assessed by SIEDY) and objective (as assessed by PDU) ED and by greater somatised anxiety than the rest of the sample. The prevalence of overt hypogonadism (total testosterone <8 nM) was significantly higher in patients with MS. Among MS components, waist circumference and hyperglycaemia were the best predictors of hypogonadism. Hypogonadal patients with MS showed higher gonadotropin and lower free testosterone levels, suggesting a primary hypogonadism. Among patients with MS, hypogonadism was present in 11.9% and 3.8% in the rest of the sample (p<0.0001) and was associated with typical hypogonadism-related symptoms, such as hypoactive sexual desire, low frequency of sexual intercourse, and depressive symptoms. CONCLUSIONS: Our data suggest that MS is associated with a more severe ED and induces somatisation. Furthermore, MS is associated with a higher prevalence of hypogonadism in patients with SD. The presence of hypogonadism can further exacerbate the MS-associated sexual dysfunction, adding the typical hypogonadism-related symptoms (including HSD, 66.7%). Recognising MS associated with hypogonadism is important for both sexual and general health and its serious potential associated risks.

Psychobiological correlates of delayed ejaculation in male patients with sexual dysfunctions.

The pathogenesis of delayed ejaculation (DE) is rather unknown, though the contribution of various psychological, marital, hormonal, and neurological factors has been advocated. In this study we systematically investigated the relative relevance of the aforementioned factors in 1,632 men who were seeking medical help for sexual dysfunction. The severity of DE was classified according to Kaplan criteria. Mild and moderate forms of DE (MMDE) recognized different risk factors than the most severe ones (anejaculation or severe DE [ASDE]). ASDE was essentially coupled with the presence of neurological diseases or with the use of serotoninergic drugs. Serotoninergic drugs also significantly increase (by at least 10-fold) the risk for MMDE, which, however, was also coupled with other relational factors (eg, partner's impaired climax, patient's hypoactive sexual desire [HSD]) or intrapsychic factors (eg, stress at work). At multiple regression analysis, some organic pathological conditions (such as psychiatric disorders and hypogonadism) were also associated with MMDE. In particular, hypogonadism retained significance for DE even after adjustment for HSD (adjusted odds ratio = 2.08 [1.11-3.89]; P < .05), suggesting other effects of testosterone deficiency on the ejaculatory reflex besides reduced libido. In conclusion, the present study demonstrates that multiple psychobiological determinants are associated with DE, a still obscure condition that substantially impairs psychosexual equilibrium of the couple.

Corticotroph adenoma of the pituitary in a patient with X-linked adrenal hypoplasia congenita due to a novel mutation of the DAX-1 gene.

OBJECTIVE: Mutations in the DAX-1 gene result in X-linked congenital adrenal hypoplasia. The classic clinical presentation is primary adrenal insufficiency in early life and hypogonadotropic hypogonadism at the time of expected puberty, but recent data have expanded the phenotypic spectrum of DAX-1 mutations. We report the occurrence of an ACTH-secreting adenoma in a patient with X-linked congenital adrenal hypoplasia. DESIGN AND METHODS: Detailed clinical, radiological and pathological investigation of the pituitary adenoma. Genomic analysis of the DAX-1 gene in the patient and his mother. RESULTS: In this patient, primary adrenal failure had been diagnosed at 3 years of age and, despite replacement therapy, at 30 years of age progressive pigmentation developed and impairment of the visual field followed. ACTH was 24 980 pg/ml and nuclear magnetic resonance disclosed a huge pituitary adenoma. Three transsphenoidal operations and radiotherapy were necessary to remove the tumor mass and control ACTH secretion. Histologically, the adenoma was composed of chromophobic and basophilic neoplastic cells with positive immunostaining for ACTH. Moreover, a novel mutation was found both in the patient and his mother: a 4 bp insertion (AGCG) at nucleotide 259, in exon 1 resulting in a frame shift and premature termination. CONCLUSIONS: This case suggests that in adrenal hypoplasia congenita the development of a pituitary adenoma should be considered when a sudden rise of ACTH occurs despite adequate steroid substitution.