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The main protease (M Pro ) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target M Pro 's catalytic cysteine. Our characterization identified potent M Pro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC 50 value of 10 nM against SARS-CoV-2 infection in ACE2 + A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent M Pro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Pathogen leak from a high-containment laboratory seriously threatens human safety, animal welfare, and environmental security. Transportation of pathogens from a higher (BSL4 or BSL3) to a lower (BSL2) containment laboratory for downstream experimentation requires complete pathogen inactivation. Validation of pathogen inactivation is necessary to ensure safety during transportation. This study established a validation strategy for virus inactivation., Methods: SARS-CoV-2 wild type, delta, and omicron variants underwent heat treatment at 95°C for 10 minutes using either a hot water bath or a thermocycler. To validate the inactivation process, heat-treated viruses, and untreated control samples were incubated with A549-hACE2 and Vero E6-TMPRSS2-T2A-ACE2 cells. The cells were monitored for up to 72 hours for any cytopathic effects, visually and under a microscope, and for virus genome replication via RT-qPCR. The quality of post-treated samples was assessed for suitability in downstream molecular testing applications., Results: Heat treatment at 95°C for 10 minutes effectively inactivated SARS-CoV-2 variants. The absence of cytopathic effects, coupled with the inability of virus genome replication, validated the efficacy of the inactivation process. Furthermore, the heat-treated samples proved to be qualified for COVID-19 antigen testing, RT-qPCR, and whole-genome sequencing., Discussion: By ensuring the safety of sample transportation for downstream experimentation, this validation approach enhances biosecurity measures. Considerations for potential limitations, comparisons with existing inactivation methods, and broader implications of the findings are discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chaki, Kahl-McDonagh, Neuman and Zuelke.)

Improved methods are needed to prevent wildlife deaths from anthrax. Caused by Bacillus anthracis , naturally occurring outbreaks of anthrax are frequent but unpredictable. The commercially available veterinary vaccine is labeled for subcutaneous injection and is impractical for large-scale wildlife vaccination programs; therefore, oral vaccination is the most realistic method to control and prevent these outbreaks. We reported the induction of an anthrax-specific lethal toxin (LeTx) neutralizing antibody response in mice following oral vaccination with alginate microcapsules containing B. anthracis Sterne strain 34F2 spores, coated with poly-L-lysine (PLL) and vitelline protein B (VpB). We continued evaluating our novel vaccine formulation through this proof-of-concept study in white-tailed deer (WTD; Odocoileus virginianus ; n = 9). We orally vaccinated WTD via needle-free syringe with three formulations of the encapsulated vaccine: 1) PLL-VpB-coated microcapsules with 10 7-8 spores/ml ( n = 5), 2) PLL-VpB-coated microcapsules with 10 9-10 spores/ml ( n = 2), and 3) PLL-coated microcapsules with 10 9-10 spores/ml ( n = 2). Although the limited sample sizes require continued experimentation, we observed an anthrax-specific antibody response in WTD serum following oral vaccination with PLL-coated microcapsules containing 10 9 spores/ ml. Furthermore, this antibody response neutralized anthrax LeTx in vitro, suggesting that continued development of this vaccine may allow for realistic wildlife anthrax vaccination programs.

Outbreaks of anthrax, caused by the soilborne bacterium Bacillus anthracis, are a continuous threat to free-ranging livestock and wildlife in enzootic regions of the United States, sometimes causing mass mortalities. Injectable anthrax vaccines are commercially available for use in livestock, and although hand injection is not a cost- or time-effective long-term management plan for prevention in wildlife, it may provide a tool for managers to target selectively animals of high conservation or economic value. Vaccine-induced anthrax-specific antibody responses have been reported previously in white-tailed deer (Odocoileus virginianus), but the protective nature was not determined. In this study, five white-tailed deer were subcutaneously vaccinated with one dose (1 mL) of the Anthrax Spore Vaccine. Eight blood collections by jugular venipuncture were conducted over 146 d to measure the anthrax-specific antibody response in each deer's serum over time. Antibodies were first detected by ELISA and later with toxin neutralization assays to estimate in vitro protection. Average peak absorbance by ELISA occurred at 14 d postvaccination, whereas average peak in vitro protection occurred at 28 d postvaccination. Observed in vitro protection on average for white-tailed deer after this single-dose vaccination protocol lasted 42-56 d postvaccination, although three individuals still maintained lethal toxin-neutralizing serum antibody titers out to 112 d postvaccination. Vaccination responses were variable but effective to some degree in all white-tailed deer., (© Wildlife Disease Association 2024.)

Earlier studies with montelukast (M) and telmisartan (T) have revealed their potential antiviral properties against SARS-CoV-2 wild-type (WT) but have not assessed their efficacy against emerging Variants of Concern (VOCs) such as Omicron. Our research fills this gap by investigating these drugs' impact on VOCs, a topic that current scientific literature has largely overlooked. We employed computational methodologies, including molecular mechanics and machine learning tools, to identify drugs that could potentially disrupt the SARS-CoV-2 spike RBD-ACE2 protein interaction. This led to the identification of two FDA-approved small molecule drugs, M and T, conventionally used for treating asthma and hypertension, respectively. Our study presents an additional potential use for these drugs as antivirals. Our results show that both M and T can inhibit not only the WT SARS-CoV-2 but also, in the case of M, the Omicron variant, without reaching cytotoxic concentrations. This novel finding fills an existing gap in the literature and introduces the possibility of repurposing these drugs for SARS-CoV-2 VOCs, an essential step in responding to the evolving global pandemic.

Brucellosis is a zoonosis caused by Brucella; B. melitensis is the most prevalent species in goats and humans. Previously, three B. melitensis peptides, rBtuB-Hia-FlgK showed antigen-specific immune responses in rodent models. The goal of this study was to evaluate the goat Th1/Th2 immune response to B. melitensis peptides. Twenty-eight animals were separated into four groups and were immunized with the rBtuB-Hia-FlgK peptides cocktail, adjuvant, PBS and Rev-1 vaccine, respectively. Peripheral blood samples were collected on days 0, 15, and 80 post-inoculation. The CD4+ and CD8+ T cells proliferation, and cytokine production of the Th-1 (IL-2, IL-12, TNF-α, and IFN-γ) and Th-2 profiles (IL-4, IL-5, and IL-10) were evaluated. An increase of CD4+/CD8+ at 15 days post-vaccination was observed and continued until the 80th. In addition, the IFN-γ, TNF-α, and IL-2 mRNA expression were typically induced by the 15th day, but only IFN-γ levels were observed at day 80 post-immunization. Brucella pathogenesis is distinguished by the presence of a large amount of Th-1 cytokines. Although a reduced amount of IFN-γ in the culture supernatant was accurately detected compared with Rev-1 after 15 days, it could be influenced by the sampling schedule, as a higher cytokine production might be induced as early as the first-week post-vaccination. The results indicate that rBtuB-Hia-FlgK induced an immune response similar to the Rev-1 vaccine. The possible use of inert molecules with the unique ability to typically induce cellular response similar to attenuated vaccine represents an attractive option that should not be ruled out., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.

As an essential enzyme of SARS-CoV-2, main protease (M Pro ) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M Pro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of M Pro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular M Pro inhibition information to assess an M Pro inhibitor. We used this assay to analyze 30 known M Pro inhibitors. Contrary to their strong antiviral effects and up to 10 μM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular M Pro inhibition potency implicating their roles in interfering with key steps other than just the M Pro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular M Pro inhibition IC 50 value of 31 nM that matches closely to its strong antiviral effect with an EC 50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

To better understand the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant lineage distribution in a college campus population, we carried out viral genome surveillance over a 7-week period from January to March 2021. Among the sequences were three novel viral variants: BV-1 with a B.1.1.7/20I genetic background and an additional spike mutation Q493R, associated with a mild but longer-than-usual COVID-19 case in a college-age person, BV-2 with a T478K mutation on a 20B genetic background, and BV-3, an apparent recombinant lineage. This work highlights the potential of an undervaccinated younger population as a reservoir for the spread and generation of novel variants. This also demonstrates the value of whole genome sequencing as a routine disease surveillance tool.

Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log 10 of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis., (© 2020 The Societies and John Wiley & Sons Australia, Ltd.)

Reproductive failure is the hallmark of brucellosis in animals. An uncommon but important complication in pregnant women who become acutely infected with Brucella melitensis is spontaneous pregnancy loss or vertical transmission to the fetus. Unfortunately, the mechanism behind reproductive failure is still obscure, partially due to the lack of a proper study model. Recently, it was demonstrated that intratracheal (IT) inoculation of nonpregnant guinea pigs would replicate features of clinical disease in humans. To determine if IT inoculation would induce reproductive disease, guinea pigs were infected at mid-gestation and monitored daily for fever and abortions. Fever developed between day 14 to 18 postinoculation, and by 3 weeks postinoculation, 75% of pregnant guinea pigs experienced stillbirths or spontaneous abortions mimicking natural disease. Next, to investigate the guinea pig as a model for evaluating vaccine efficacy during pregnancy, nonpregnant guinea pigs were vaccinated with S19, 16MΔ vjbR + Quil-A, or 100 μl PBS + Quil-A (as control). Guinea pigs were bred and vaccinated guinea pigs were challenged at mid-gestation with B. melitensis IT inoculation and monitored for fever and abortions. Vaccination with both vaccines prevented fever and protected against abortion. Together, this study indicates that pregnant guinea pigs are an appropriate animal model to study reproductive disease and offer an improved model to evaluate the ability of vaccine candidates to protect against a serious manifestation of disease., (Copyright © 2020 American Society for Microbiology.)

An oral vaccine against anthrax ( Bacillus anthracis ) is urgently needed to prevent annual anthrax outbreaks that are causing catastrophic losses in free-ranging livestock and wildlife worldwide. The Sterne vaccine, the current injectable livestock vaccine, is a suspension of live attenuated B. anthracis Sterne strain 34F2 spores (Sterne spores) in saponin. It is not effective when administered orally and individual subcutaneous injections are not a practical method of vaccination for wildlife. In this study, we report the development of a microencapsulated oral vaccine against anthrax. Evaluating Sterne spore stability at varying pH's in vitro revealed that spore exposure to pH 2 results in spore death, confirming that protection from the gastric environment is of main concern when producing an oral vaccine. Therefore, Sterne spores were encapsulated in alginate and coated with a protein shell containing poly-L-lysine (PLL) and vitelline protein B (VpB), a non-immunogenic, proteolysis resistant protein isolated from Fasciola hepatica . Capsule exposure to pH 2 demonstrated enhanced acid gel character suggesting that alginate microcapsules provided the necessary protection for spores to survive the gastric environment. Post vaccination IgG levels in BALBc/J mouse serum samples indicated that encapsulated spores induced anti-anthrax specific responses in both the subcutaneous and the oral vaccination groups. Furthermore, the antibody responses from both vaccination routes were protective against anthrax lethal toxin in vitro, suggesting that further optimization of this vaccine formulation may result in a reliable oral vaccine that will conveniently and effectively prevent anthrax in wildlife populations., Competing Interests: Competing interestsA.C.R-.F. is a managing partner of NanoRelease Technologies LLC (NRT), a San Antonio-based company specializing in vaccine delivery platforms. The terms of this arrangement have been reviewed and approved by TXAgriLife Research and Texas A&M University in accordance with their conflict of interest policies., (© The Author(s) 2020.)

Brucella canis is a Gram-negative, facultative intracellular bacterium and the causative agent of canine brucellosis, a highly contagious disease of dogs that can be transmitted to humans. Unfortunately, no vaccine is available to prevent infection. We recently characterized the kinetics of B. canis infection in the mouse model, establishing the required dose necessary to achieve systemic infection. The objective of this study was to investigate the utility of the mouse model in assessing canine brucellosis vaccine candidates and to subsequently investigate the safety and efficacy of a live attenuated vaccine, the B. canis RM6/66 Δ vjbR strain. Mice vaccinated with a dose of 10 9 CFU of the vaccine strain by both intraperitoneal and subcutaneous routes were afforded significant protection against organ colonization and development of histopathologic lesions following intraperitoneal challenge. Addition of an adjuvant or a booster dose 2 weeks following initial vaccination did not alter protection levels. Vaccination also resulted in a robust humoral immune response in mice, and B. canis RM6/66 Δ vjbR was capable of activating canine dendritic cells in vitro These data demonstrate that the B. canis RM6/66 Δ vjbR strain shows promise as a vaccine for canine brucellosis and validates the mouse model for future vaccine efficacy studies. IMPORTANCE Canine brucellosis, caused by Brucella canis , is the primary cause of reproductive failure in dogs and represents a public health concern due to its zoonotic nature. Cases in dogs in the United States have been increasing due to the persistent nature of the bacterium, deficiencies in current diagnostic testing, and, most importantly, the lack of a protective vaccine. Current estimates place the seroprevalence of B. canis in the southern United States at 7% to 8%, but with the unprecedented rates of animals moving across state and international borders and the lack of federal regulations in regard to testing, the true seroprevalence of B. canis in the United States may very well be higher. Vaccination represents the most effective method of brucellosis control and, in response to the demand for a vaccine against B. canis , we have developed the live attenuated B. canis RM6/66 Δ vjbR vaccine strain capable of protecting mice against challenge., (Copyright © 2020 Stranahan et al.)

As a natural host species for Brucella melitensis , pregnant sheep offer an ideal model to evaluate vaccine candidates for safety. B. melitensis strain Rev. 1 has been used almost exclusively to prevent brucellosis in small ruminants, but it causes abortions when given to pregnant animals. To evaluate the comparative safety of the candidate Brucella melitensis 16M ΔvjbR , pregnant sheep ( n  = 6) were vaccinated subcutaneously with 1 × 10 10 CFU/ml of 16M ΔvjbR or 1 × 10 9 CFU/ml Rev. 1 at a highly susceptible stage of gestation (approximately 70 days). 16M ΔvjbR resulted in only 1 abortion (1 of 6) compared with 4 of 6 (66.7%) abortions in the Rev. 1 cohort. The placenta was evaluated by culture to determine if vaccination resulted in colonization. As another measure of safety, effects of B. melitensis on the fetus/offspring (vertical transmission) was evaluated by culture and histopathology of fetal tissues to determine if vaccination prevented infection of the fetus. Vaccination with 16M ΔvjbR resulted in less vertical transmission than Rev. 1. To determine if vaccination was efficacious and could reduce tissue colonization in sheep, the same cohort of sheep were challenged 5 weeks postpartum by conjunctival inoculation with 1 × 10 7 CFU/ml B. melitensis Protection was similar between Rev. 1 and 16M ΔvjbR , with no statistical difference in colonization in the target organs. Overall, the 16M ΔvjbR vaccine was considered safer than Rev. 1 based on a reduced number of abortions and limited infection in the offspring. Future experiments are needed to further refine the vaccine dose to increase the safety margin and to evaluate protection in pregnant ewes. IMPORTANCE Brucellosis is one of the most commonly reported zoonotic disease with a worldwide distribution. Of the 12 Brucella species, Brucella melitensis is considered the most virulent and causes reproductive failure (abortions/stillbirths) in small ruminants, which can spread the disease to other animals or to humans. Vaccination of small ruminants is a key measure used to protect both human and animal health. However, the commercially available live-attenuated vaccine for Brucella melitensis Rev. 1 retains virulence and can cause disease in animals and humans. In order to evaluate the safety and efficacy in sheep, we vaccinated pregnant sheep with 16M ΔvjbR Our results indicate that 16M ΔvjbR was safer for use during pregnancy, provided a similar level of protection as Rev. 1, and could be considered an improved candidate for future vaccine trials., (Copyright © 2020 Hensel et al.)

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19 vjbR and B. abortus ΔvirB2 While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19 vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety., (Copyright © 2020 American Society for Microbiology.)

Background: Podosomes are membrane-bound adhesive structures formed by actin remodeling. They are capable of extracellular matrix (ECM) degradation, which is a prerequisite for cancer cell invasion and metastasis. The signaling mechanism of podosome formation is still unknown in cancer. We previously reported that Nck adaptors regulate directional cell migration and endothelial lumen formation by actin remodeling, while deficiency of Nck reduces cancer metastasis. This study evaluated the role of Nck adaptors in podosome biogenesis and cancer invasion., Methods: This study was conducted in vitro using both healthy cells (Human Umbilical Vein Endothelial Cell, 3T3 fibroblasts) and cancer cells (prostate cancer cell line; PC3, breast cancer cell line; MDA-MB-231). Confocal and TIRF imaging of cells expressing Green Fluorescence Protein (GFP)  mutant under altered levels of Nck or downstream of kinase 1 (Dok1) was used to evaluate the podosome formation and fluorescent gelatin matrix degradation. Levels of Nck in human breast carcinoma tissue sections were detected by immune histochemistry using Nck polyclonal antibody. Biochemical interaction of Nck/Dok1 was detected in podosome forming cells using immune precipitation and far-western blotting., Results: This study demonstrates that ectopic expression of Nck1 and Nck2 can induce the endothelial podosome formation in vitro. Nck silencing by short-hairpin RNA blocked podosome biogenesis and ECM degradation in cSrc-Y530F transformed endothelial cells in this study. Immunohistochemical analysis revealed the Nck overexpression in human breast carcinoma tissue sections. Immunoprecipitation and far-western blotting revealed the biochemical interaction of Nck/p62Dok in podosome forming cells., Conclusions: Nck adaptors in interaction with Dok1 induce podosome biogenesis and ECM degradation facilitating cancer cell invasion, and therefore a bona fide target of cancer therapy., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Brucellosis in swine is caused by Brucella suis, a bacterial infection of nearly worldwide distribution. Brucella suis is also transmissible to humans, dogs and cattle and is considered a reemerging disease of public health concern. To date, there is no effective vaccine for swine. This prompted us to investigate the potential use of the commercially available vaccine for cattle or the live attenuated vaccine candidate S19Δ vjbR . As the first step, we sought to study the safety of the vaccine candidates when administered in pregnant sows, since one of the major drawbacks associated with vaccination using Live Attenuated Vaccines (LAV) is the induction of abortions when administered in pregnant animals. Fifteen pregnant gilts at mid-gestation were divided into four groups and subsequently vaccinated subcutaneously using different formulations containing 2.0 ± 0.508 × 10 9  CFU of either S19 or S19Δ vjbR . Vaccination in pregnant animals with the vaccine candidates did not induce abortion, stillbirths or a reduction in litter size. Multiple tissues in the gilts and piglets were examined at the time of delivery to assess bacterial colonization and histopathological changes. There was no evidence of vaccine persistence in the gilts or bacterial colonization in the fetuses. Altogether, these data suggest that both vaccine candidates are safe for use in pregnant swine. Analysis of the humoral responses, specifically anti- Brucella IgG levels measured in serum, demonstrated a robust response induced by either vaccine, but of shorter duration (4-6 weeks post-inoculation) compared to that observed in cattle or experimentally infected mice. Such a transient humoral response may prove to be beneficial in cases where the vaccine is used in eradication campaigns and in the differentiation of vaccinated from infected animals. This study provides evidence to support future efficacy studies of both vaccine candidates in swine., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Allison Rice-Ficht, managing partner of NanoRelease Technologies (NRT), LLC Inc., has a 95% equity interest in NRT, a company involved in vaccine delivery platforms. The terms of this arrangement have been reviewed and approved by TXAgriLife Research and Texas A&M University in accordance with their conflict of interest policies.

Osteoarticular brucellosis is the most common complication in Brucella -infected humans regardless of age, sex, or immune status. The mechanism of bone destruction caused by Brucella species remained partially unknown due to the lack of a suitable animal model. Here, to study this complication, we explored the suitability of the use of the NOD- scid IL2rγ null mouse to study osteoarticular brucellosis and examined the potential use of this strain to evaluate the safety of live attenuated vaccine candidates. Mice were inoculated intraperitoneally with a single dose of 1 × 10 4 , 1 × 10 5 , or 1 × 10 6 CFU of B. abortus S19 or the vaccine candidate B. abortus S19 ΔvjbR and monitored for the development of side effects, including osteoarticular disease, for 13 weeks. Decreased body temperature, weight loss, splenomegaly, and deformation of the tails were observed in mice inoculated with B. abortus S19 but not in those inoculated with S19 ΔvjbR Histologically, all S19-inoculated mice had a severe dose-dependent inflammatory response in multiple organs. The inflammatory response at the tail was characterized by the recruitment of large numbers of neutrophils, macrophages, and osteoclasts with marked bone destruction. These lesions histologically resembled what is typically observed in Brucella -infected patients. In contrast, mice inoculated with B. abortus S19 ΔvjbR did not show significant bone changes. Immunofluorescence, in situ hybridization, and confocal imaging demonstrated the presence of Brucella at the sites of inflammation, both intra- and extracellularly, and large numbers of bacteria were observed within mature osteoclasts. These results demonstrate the potential use of the NOD- scid IL2rγ null mouse model to evaluate vaccine safety and further study osteoarticular brucellosis., (Copyright © 2019 American Society for Microbiology.)

B. melitensis is considered the most virulent of the Brucella species, and a need exists for an improved laboratory animal model of infection that mimics natural transmission and disease. Guinea pigs are highly susceptible to infection with Brucella spp. and develop a disease syndrome that mimics natural disease after aerosol inoculation. Intratracheal inoculation is a targeted means of generating aerosols that offer advantages over aerosol chamber delivery. To establish this delivery method, female, Hartley guinea pigs were infected via intratracheal inoculation with PBS or 16M B. melitensis at low dose (101 to 103) or high dose (106 to 108) and monitored for 30 days for signs of disease. Guinea pigs in the high dose groups developed fever between 12-17 days post-inoculation. Bacteria were recovered from the spleen, liver, lymph nodes, lung, and uterus at 30-days post-inoculation and demonstrated dose dependent mean increases in colonization and pathologic changes consistent with human brucellosis. To study the kinetics of extrapulmonary dissemination, guinea pigs were inoculated with 107 CFU and euthanized at 2-hours post inoculation and at weekly intervals for 3 weeks. 5.8x105 to 4.2x106 CFU were recovered from the lung 2 hours post-inoculation indicating intratracheal inoculation is an efficient means of infecting guinea pigs. Starting at 1-week post inoculation bacteria were recovered from the aforementioned organs with time dependent mean increases in colonization. This data demonstrates that guinea pigs develop a disease syndrome that models the human manifestation of brucellosis, which makes the guinea pig a valuable model for pathogenesis studies., Competing Interests: The authors have declared that no competing interests exist.

Although it is known that noncatalytic region of tyrosine kinase (Nck) regulates cell adhesion and migration by bridging tyrosine phosphorylation with cytoskeletal remodeling, the role of Nck in tumorigenesis and metastasis has remained undetermined. Here we report that Nck is required for the growth and vascularization of primary tumors and lung metastases in a breast cancer xenograft model as well as extravasation following injection of carcinoma cells into the tail vein. We provide evidence that Nck directs the polarization of cell-matrix interactions for efficient migration in three-dimensional microenvironments. We show that Nck advances breast carcinoma cell invasion by regulating actin dynamics at invadopodia and enhancing focalized extracellular matrix proteolysis by directing the delivery and accumulation of MMP14 at the cell surface. We find that Nck-dependent cytoskeletal changes are mechanistically linked to enhanced RhoA but restricted spatiotemporal activation of Cdc42. Using a combination of protein silencing and forced expression of wild-type/constitutively active variants, we provide evidence that Nck is an upstream regulator of RhoA-dependent, MMP14-mediated breast carcinoma cell invasion. By identifying Nck as an important driver of breast carcinoma progression and metastasis, these results lay the groundwork for future studies assessing the therapeutic potential of targeting Nck in aggressive cancers., (© 2017 Morris et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose of Review: Brucellosis is a neglected, zoonotic disease of nearly worldwide distribution. Despite brucellosis being recognized as a reproductive disease in animals, it has been historically known as a flu-like illness in humans with little or no significant role in maternal or newborn health. This review focuses on what is currently known relative to the epidemiology of brucellosis in human pregnancy as well as new insights of placental immunology., Recent Findings: New evidence suggests that maternal infection poses a significant risk factor for adverse pregnancy outcomes including increased risk for miscarriage during the first and second trimester of gestation, preterm delivery, and vertical transmission to the fetus. Adverse pregnancy outcomes were not associated with any specific clinical sign. However, prompt diagnosis and treatment significantly decreased the risk of miscarriage or any other adverse effect., Summary: Brucellosis during pregnancy should be considered a significant risk factor for adverse pregnancy outcomes in humans. The identification of the mechanism behind bacterial tropism should prove powerful for the development of new countermeasures to prevent these detrimental effects. Increased awareness concerning brucellosis in pregnant women, its transmission, and prevention measures should be considered as a pressing need., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interest.

Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy., (© 2015 Chaki et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The crosstalk between cells and their microenvironment enables cellular adaptation to external mechanical cues through the remodeling of cytoskeletal structures and cell-matrix adhesions to ensure normal cell function. This study investigates the relationship between the cytoskeletal tension and integrin α5β1 adhesion strength to the matrix (i.e. fibronectin) in the context of RhoA-Src crosstalk. Integration of atomic force microscopy (AFM) with total internal reflection fluorescence and spinning-disk confocal microscopy enabled acquisition of complementary structural and functional measurements on live vascular smooth muscle cells expressing RhoA and c-Src variants (wild-type, dominant negative, constitutively active). Single ligand-receptor interaction measurements performed with AFM probes functionalized with fibronectin showed that RhoA and c-Src activation have different effects on cytoskeletal tension development, inducing two distinct force-stiffness functional regimes for α5β1-integrin binding to fibronectin. Moreover, fluorescence measurements showed that c-Src activation had a modest effect on actin morphology, while RhoA significantly modulated stress fiber formation. In addition, c-Src was associated with regulation of myosin light chain (MLC) phosphorylation, suggesting a c-Src-dependent modulation of RhoA pathway through activation of downstream effectors. Therefore, c-Src may be a possible component of cytoskeletal tension regulation through MLC activation. Our findings suggest that Src and RhoA coordinate a regulatory network that determines cytoskeletal tension through activation of actomyosin contractility. In turn, the cytoskeletal tension state modulates integrin α5β1-fibronectin adhesion force.

Planar and apical-basal cellular polarization of epithelia and endothelia are crucial during morphogenesis. The establishment of these distinct polarity states and their transitions are regulated by signaling networks that include polarity complexes, Rho GTPases, and phosphoinositides. The spatiotemporal coordination of signaling by these molecules modulates cytoskeletal remodeling and vesicle trafficking to specify membrane domains, a prerequisite for the organization of tissues and organs. Here we present an overview of how activation of the WASp/Arp2/3 pathway of actin remodeling by Nck coordinates directional cell migration and speculate on its role as a signaling integrator in the coordination of cellular processes involved in endothelial cell polarity and vascular lumen formation.

Directional migration requires the coordination of cytoskeletal changes essential for cell polarization and adhesion turnover. Extracellular signals that alter tyrosine phosphorylation drive directional migration by inducing reorganization of the actin cytoskeleton. It is recognized that Nck is an important link between tyrosine phosphorylation and actin dynamics; however, the role of Nck in cytoskeletal remodeling during directional migration and the underlying molecular mechanisms remain largely undetermined. In this study, a combination of molecular genetics and quantitative live cell microscopy was used to show that Nck is essential in the establishment of front-back polarity and directional migration of endothelial cells. Time-lapse differential interference contrast and total internal reflection fluorescence microscopy showed that Nck couples the formation of polarized membrane protrusions with their stabilization through the assembly and maturation of cell-substratum adhesions. Measurements by atomic force microscopy showed that Nck also modulates integrin α5β1-fibronectin adhesion force and cell stiffness. Fluorescence resonance energy transfer imaging revealed that Nck depletion results in delocalized and increased activity of Cdc42 and Rac. By contrast, the activity of RhoA and myosin II phosphorylation were reduced by Nck knockdown. Thus, this study identifies Nck as a key coordinator of cytoskeletal changes that enable cell polarization and directional migration, which are crucial processes in development and disease.

Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200 x g for 30 min through a 40% Equipure silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 microg of total RNA per 100 x 10(6) sperm. To evaluate RNA quantity and quality, we used a NanoDrop spectrophotometer and Agilent Bioanalyzer. A protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed to determine sperm RNA contamination with genomic DNA or RNA from somatic cells. By these methods, hybridization- and sequencing-quality RNA was isolated from 11 samples of stallion sperm. Stallion testis biopsy with a 14 gauge 22 mm deep biopsy needle yielded approximately 12 microg of good quality total RNA, and could serve as an alternative to excision surgery for sample procurement. Compared to RNA isolation from testis, the sperm required advanced processing and RNA quality control. The described methodologies provided a foundation to establish functional genomic studies of stallion fertility., ((c) 2010 Elsevier Inc. All rights reserved.)

Background: The study was conducted to test the potential of using dienogest (DNG) plus testosterone undecanoate (TU) in rats for development of a once-a-month injectable male hormonal contraceptive., Study Design: Dose selection studies were initiated with administration of DNG in three different doses of 20, 30 and 40 mg/kg body weight (bw) per week plus TU 25 mg/kg bw once in every 6 weeks. Status of spermatogenesis and sperm count in epididymis was evaluated. The frequency of DNG intervention was later extended to every 2- and 4-week intervals. Mating studies, toxicity and reversibility of spermatogenesis following stoppage of treatment were carried out with DNG 40 mg/kg bw at 4-week intervals., Results: Complete arrest of spermatogenesis was observed after 60 days of treatment at all doses of DNG (20, 30 and 40 mg/kg bw per week)+TU. However, weights of testis and accessory sex organs (epididymis, prostate and seminal vesicle) declined significantly 60 days post treatment compared to vehicle-treated controls. Epididymis in the treated animals was completely devoid of sperm. When the frequency of DNG injection (20 mg/kg bw) was extended to once every 15 days, a few immotile and decapitated sperm were observed in the epididymis. With TU treatment unchanged, animals receiving DNG (40 mg/kg bw) once either every 2- or 4-week intervals demonstrated good and uniform arrest of spermatogenesis. DNG 40 mg/kg per 4 weeks+TU also demonstrated a significant rise in germ cell apoptosis in the seminiferous epithelium. There was no significant increase in the serum high-density lipoprotein and low-density lipoprotein levels at the end of 120 days of treatment. Following withdrawal of treatment after 60 or 120 days, qualitative restoration of spermatogenesis was rapid in the former compared to the latter., Conclusion: Dienogest plus TU has the potential for development as a monthly injectable showing reversible hormonal male contraception with good efficacy.

Objective: To determine whether or not sperm function parameters are altered in male partners of couples with a history of idiopathic recurrent pregnancy loss (RPL)., Design: In comparison with proven fertile volunteers, sperm function parameters like hypo-osmotic swelling (HOS), acrosomal status (AS), and nuclear chromatin decondensation (NCD) were assessed in vitro from male partners of couples with a history of idiopathic RPL., Setting: Infertility clinic and andrology laboratory at National Institute of Health and Family Welfare., Patient(s): Male partners of couples with a history of idiopathic RPL and proven fertile male volunteers (control)., Intervention(s): Standard semen analysis, assessment of sperm morphology, and sperm function with tests such as HOS, AS, and NCD., Main Outcome Measure(s): Sperm paameters, such as HOS, AS, and NCD, were assessed in semen samples from RPL in comparison with the proven fertile control group., Result(s): Semen samples from the idiopathic RPL group showed below normal test scores in 57.1% of the cases for all three sperm parameters. The highest aberration (83% of cases) in sperm attributes was observed in NCD, followed by AS (45.7%) and HOS (42.9%). In contrast, abnormality in sperm morphology was limited to 5.7% of the cases. Subnormal sperm function is directly proportional with subnormal sperm motility (<50%) in 23% of the cases. Even in semen samples with normal sperm motility, sperm function scores were below normal in 31.4% of the RPL group., Conclusion(s): Reduction in test scores of sperm function, like HOS, AS, and NCD, in male partners of couples with idiopathic RPL suggests that sperms with altered or lowered functional competencies, if they fertilize the oocytes, may lead to the development of an unsustainable embryo resulting in early pregnancy loss. Normal sperm motility does not always ensure normal sperm function scores.

Release of copper and its effect on functional integrity of human sperms in vitro were assessed following co-incubation of semen with CuT 380A. After 30 min of incubation with semen, release of copper ions from CuT 380A was found to be 9.2 to 40 times higher compared to control incubations with PBS. Sperm function tests, when simultaneously performed following loss of motility in sperms (> 95%) after 120 min of copper exposure, depicted a significant (P < 0.001) reduction in sperm viability and hypo-osmotic swelling (HOS) response. However, the affected sperm populations revealed no significant alterations in other functional tests like acrosomal status or nuclear chromatin decondensation. It is therefore concluded that the high release of copper from CuT 380A drastically lowers sperm motility, viability and HOS response but only marginally affects the acrosome status or nuclear chromatin condensation in short term incubations.

Objective: To develop a rapid, sensitive, and reproducible hypoosmotic swelling test kit for the assessment of plasma membrane integrity of human sperm in vitro., Design: Prospective comparison of results with the World Health Organization (WHO) method, performed simultaneously., Setting: Infertility center in a major city in India., Patient(s): Couples who presented for infertility evaluation., Intervention(s): The sperm tail-coiling pattern representing sperm plasma membrane integrity was analyzed by using different concentrations of NaCl and also with plain double distilled water (ddH(2)O). Hypoosmotic swelling solution with 2% NaCl in ddH(2)O equivalent to 68 Osm/L was selected for further analysis because it provided the highest and the qualitatively best type (g) of tail coiling among the various other options tried in comparison., Main Outcome Measure(s): A rapid laboratory test kit for the assessment of plasma membrane integrity of sperm was developed that is equally sensitive and reproducible as that described in the WHO protocol. The test was validated by using different normal and subnormal semen samples and in comparison with the standard WHO protocol., Result(s): After the screening with different hypoosmotic solutions by using semen samples from fertile volunteers, NaCl (2%) provided the highest and the best types of typical tail coiling, characteristic of sperm with good plasma membrane integrity. The study was then extended to 60 normozoospermic semen samples, which demonstrated a hypoosmotic swelling response of 69.5% +/- 5.23% and 71.5% +/- 4.89%, as per WHO or the present modified method, respectively (coefficient of correlation, r = 0.741). In 13 oligospermic and 18 teratozoospermic subjects, the hypoosmotic swelling response of sperm as per the new method were 36.4% +/- 5.75% and 34.2% +/- 7.78%, respectively, which were comparable to those obtained through the WHO method. There was a 15% drop in viability of sperm after the hypoosmotic challenge (5 min), irrespective of the hypoosmotic solution used. No other morphological alterations in sperm were observed after the hypoosmotic challenge., Conclusion(s): The new test kit can be used routinely in laboratories for assessment of plasma membrane integrity of sperm in vitro.

The clinical significance of exogenous hCG treatment is to stimulate steroidogenesis and spermatogenesis in the testis. However, the pathogenesis of detrimental effects on the testis arising out of chronic hCG treatment is yet to be clearly ascertained. In the present study we have shown that hCG treatment (100 IU/day) to rats for 30 days raises testicular oxidative stress leading to germ cell apoptosis and impairment of spermatogenesis. The treatment raises testicular H(2)O(2) levels along with increase in lipid peroxidation and concomitant decrease in the enzymatic antioxidant activities like superoxide dismutase, catalase and glutathione-s-transferase. The rise in the number of apoptotic germ cells was associated with up regulation of Fas protein expression and caspase-3 activity in the testis. However, serum testosterone which was elevated by 15 days of hCG treatment declined to pretreatment levels by 30 days. No significant alteration in serum gonadotropins was observed. The above findings indicate that the pathogenesis of deleterious effects following chronic hCG treatment is due to increase in testicular oxidative stress with high H(2)O(2) availability leading to apoptosis among germ cells.

In order to understand the pathogenesis of estradiol induced effects in the seminiferous epithelium, studies were undertaken in adult rats with estradiol-3-benzoate administered for different durations. After 30 d of treatment, a significant rise in lipid peroxidation with concomitant fall in the activities of superoxide dismutase and catalase was observed. Both, serum and intra-testicular testosterone levels were found severely depleted. Seminiferous epithelium was devoid of elongated spermatids and spermatozoa by 30 d of treatment. Number of spermatocytes and round spermatids were significantly (p < 0.001) reduced. Flowcytometric analysis confirmed a drastic reduction of the haploid cell population (1c peak). Beginning from day 10 of treatment, there was a consistent rise in the number of pyknotic/apoptotic germ cells in the seminiferous epithelium. A gradual increase in Bax protein expression was observed with the duration of treatment. The shift in Bax immunostaining from the cytoplasm and nucleus of germ cells (at 10 d of treatment) to only nuclei of cells by 30 d of treatment was also noticed. By this time testicular tissue showed three-fold increase in caspase-8 enzyme activity. Viable testicular cells isolated in vitro decreased drastically subsequent to different periods of estradiol treatment. The above findings substantiate the fact that the testicular pathogenesis of estradiol benzoate treatment may be primarily because of altered reproductive hormone levels and high oxidative stress leading to germ cell apoptosis and subsequent germ cell loss in the seminiferous epithelium.

H(2)O(2) is one of the active reactive oxygen species secreted by macrophages that are seen closely aligned with Leydig cells in the testicular interstitium. The present study was initiated to investigate the role of H(2)O(2) on Leydig cell function in vitro at physiological concentrations. Significant decrease in both testosterone production (p < 0.05) and 3 beta-hydroxysteroid dehydrogenase activity (p < 0.05) in adult Leydig cells were observed even with H(2)O(2) at low concentrations (30 - 50 microM). H(2)O(2) exposure increased oxidative stress in Leydig cells with the rise in lipid peroxidation and fall in the activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT) & glutathione-s-transferase (GST). There was also a marginal increase (approximately 8%) in cell apoptosis accompanied by rise in FasL expression and caspase-3 activation. The above findings indicate that H(2)O(2) as a bio-molecule modulates Leydig cell function at or below physiological concentrations through a variety of actions like decrease in steroidogenic enzyme activity and increase in oxidative stress and apoptosis.

Germ cell death and their removal from the seminiferous epithelium are common in the affected testis in conditions of unilateral ischemia or cryptorchidism; the similarities and differences, however, have not been studied between these two conditions. The present study was designed to examine the severity of the effect on testicular germ cells during the initial stages of both ischemia and cryptorchidism, which have significant implications on the restoration of fertility following surgical repair. Complete absence of spermatids was observed following 12 hr of ischemia as compared to 7 days of cryptorchidism. Germ cell removal in either case was in the direction of lumen to basement membrane leaving only a single layer of cells by 24 hr of unilateral ischemia as compared to 15 days of cryptorchidism. Levels of intratesticular testosterone was found lower in cryptorchidism (7 days) but not in ischemia till 24 hrs. Giant cells frequently observed in cryptorchid testis were absent in the ischemic seminiferous epithelium. There was a gradual increase in the number of apoptotic and non-viable cells; the latter was more than 95% by 24 hr of ischemia. In contrast, approximately 85% testicular cells were nonviable till 15 days of cryptorchidism. The 1c peak representing the population of haploid cells was significantly reduced in cryptorchidism (7 days), while the peak was completely abolished by 24 hr of ischemia. Rise in the levels of oxidative stress in the affected testis was observed identically during the initial stages. These findings indicate that coupled with the rise in tissue oxidative stress, the number of apoptotic/nonviable germ cells was alarmingly high (> 80%) by 15 days of cryptochidism or 24 hr of ischemia. Restoration of complete spermatogenesis following surgical repair may not be possible in such cases because of these acute adverse effects.

In order to determine that apoptosis is responsible for large-scale germ cell elimination, we analyzed cells from cryptorchid testes both in histological sections and among those isolated in vitro. Apoptotic testicular cells during 3 to 7 days were only 8 to 30%, reaching a maximum of 80% by the end of 15 days of cryptorchidism. A similar trend was also observed with the number of dead cells. The process of large-scale germ cell removal in the initial stages was facilitated by the formation of multinucleated giant cells, which stained negative for apoptosis. Increase in oxidative stress and decrease in intratesticular testosterone was also observed. The above findings indicate that large-scale germ cell removal, at least during initial stages of cryptorchidism is not solely as a result of apoptosis. Declined intra testicular testosterone, elevated temperature and high oxidative stress following cryptorchidism probably affect cell viability and trigger a fast pace cell removal through giant cell formation.

Human sperm susceptibility to oxidative stress is vital as it affects various characteristics of sperm function. In the present study, we report a simple, sensitive and quick method of assessing the capacity of the sperms to withstand increased oxidative stress. The basis for the test was derived from the fact that human sperms suspended in Ham's F-10 medium tend to lose the forward progressive motility when co-incubated with H(2)O(2) (600 microm). Replacement of the medium with seminal plasma (1: 1) was able to reduce the loss of sperm motility (40%). Retention of sperm motility in semen (0-30%) following 10 min of H(2)O(2) (600 microm) exposure was taken as the criteria for delineating the quality of sperm as poor, moderate, good and excellent types. The protocol was tested in 87 subjects presenting a normal semen profile. On the basis of this test, 44% of the semen samples were classified as poor and the rest as moderate, good or excellent. Lipid peroxidation was found higher in the sperms from the 'poor' category. Activities of superoxide dismutase and catalase were also significantly elevated in the seminal plasma of these subjects as compared with combined categories of good or excellent. The test described here can be used routinely in laboratory investigations to assess sperm susceptibility to oxidative stress in subjects presenting a normal semen profile.

Ischaemia induced germ cell apoptosis in rat testis was studied in detail to find out (i) spermatogenic stage or seminiferous epithelium region specific involvement of germ cells in apoptosis, (ii) preferential specificity of a particular germ cell type to become apoptotic and (iii) the ratio of live and dead testicular cells isolated in vitro after various period of ischaemic induction. Cell apoptosis, as observed in histological sections increased from 1 to 24 h of ischaemia. Apoptosis was not restricted to any specific germ cell type but was observed simultaneously in all the cell types in the initial hours (1-6 h) of ischaemia. No spermatogenic stage specific preference in apoptotic induction was also observed. However, as the duration of ischaemia progressed, the cell types observed to be most affected in number and morphology were the spermatids followed by spermatocytes. Centrally located tubules of testis were affected first than those located in the periphery. Overexpression of Bax staining was limited to few germ cell nuclei only. More than 95% of the germ cells in the control testis that earlier showed trypan blue dye exclusion were found stained after 12 h of ischaemia. Starting from early hours (1 h), lipid peroxidation rose proportionally with the duration of ischaemia while superoxide dismutase (SOD) and catalase activities were found decreased. Significant (p < 0.05) increase in the activities of glutathion-s-transferase and levels of hydrogen peroxide were observed after 6 h of ischaemia. These findings indicate that the physiological processes of oxidative stress have a direct linkage to the extent of germ cell apoptosis in the seminiferous epithelium.

Objectives: To examine the effects of varying doses of prepubertal human chorionic gonadotropin (hCG) administration on postpubertal germ cell status and androgen status in rats. The long-term effects of prepubertal hCG administration on postpubertal testicular function are still debated., Methods: Forty male prepubertal Wistar rats, aged 20 days, were divided into four equal groups. Group 1 served as the controls. Each rat in groups 2, 3, and 4 received subcutaneous hCG injections of 5 IU, 10 IU, and 50 IU, respectively, on days 20 and 30 of life. Serum testosterone levels were estimated on days 33 and 70. On day 70, the left testis of each rat was harvested for DNA flow cytometric analysis., Results: The testosterone levels on day 33 progressively increased from groups 1 through 4, and the difference between any two groups was statistically significant. In contrast, the testosterone levels on day 70 were greatest in group 1 and progressively decreased in groups 2 through 4, with the lowest value in group 4. On day 70, only group 4 rats had a significantly reduced haploid cell population compared with all other groups., Conclusions: Prepubertal hCG administration adversely affects testosterone levels, and a high dose of hCG has adverse effects on the germ cell haploid cell population. A critical reevaluation of the use of hCG is required in prepubertal boys, especially with respect to the dosage.

Magnetic resonance (MR) imaging is a useful tool to study the anatomy of the testis while 31P magnetic resonance spectroscopy (MRS) provides a non-invasive alternative method to demonstrate the metabolic status of testes. This study was designed to test whether the protective role of cyclosporine in experimental unilateral blunt testicular trauma (UBTT) could be assessed by 31P MRS. Male Wistar rats (n = 30) aged 20 days were randomised into group I (sham surgery), group II (UBTT) and group III (UBTT and cyclosporine for 7 days). Contralateral testicles of 5 rats from each group was evaluated by 31P MRS at 30 and 60 days of age and phosphomonoesters (PM), phosphodiesters (PD), and adenosine triphosphate (ATP) levels were measured. At 60 days of age the PM/ATP ratio was 0.32+/-0.08 in group I whereas it was 0.68+/-0.31 in the group II (p<0.05). Group III rats showed PM, PD and PM/ATP ratios similar to the controls. In conclusion, it is observed that UBTT causes contralateral testicular damage which could be prevented by short-term cyclosporine treatment and 31P MRS is an excellent modality for such an evaluation.

Following the intravasal injection of a new male contraceptive RISUG (reversible inhibition of sperm under guidance) in volunteers, routine semen analysis, semen biochemistry and germ cell morphology were evaluated in comparison with the corresponding preinjection samples for a maximum period of 6 months. Sperm counts in all 25 subjects before injection varied from 45 to 120 x 10(6)/ml. Out of 25 subjects, 6 became azoospermic after 1 month, 15 after 2 months, 3 after 3 months and 1 after 4 months of contraceptive injection. The mean volume of the ejaculates was found to be less as compared to preinjection samples. Occasional sperm or sperm heads and immature germ cells were identified in only a few postinjected subjects. However, no pregnancy was reported in these subjects during the study period. Abnormal morphology found in most of the sperm, but not in the accompanying immature germ cells, may be due to a charge-related effect on the former but not on the latter cells. Neutral alpha-glucosidase, the biochemical marker for epididymis, was estimated to be significantly lower in the seminal plasma of all the postinjected subjects. On the other hand, acid phosphatase activity and fructose levels in the seminal plasma were found to be in the normal range. Based on the above findings, it is concluded that at least for the present study period, RISUG, a new male contraceptive, is effective as a partially occluding agent in the vas deferens.

The reactive oxygen species (ROS) that is most damaging to human spermatozoa is hydrogen peroxide. Using an artificial medium Ham's F-10, we have evaluated the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on various sperm function characteristics to develop a water-based vaginal contraceptive. H(2)O(2) at 30 and 60 micro M had no effect on sperm motility. At 120 micro M of H(2)O(2), a significant reduction (90-95%) in motility was observed after 120 min. However, at 600 micro M of H(2)O(2), all the sperm were found immotile within 15 min of incubation. At much higher concentrations (1200-1500 micro M), the effect was immediate. After immobilization with H(2)O(2) (600 micro M in media), viability in sperm declined to 36% after 30 min, and was further reduced to 5% in 60 min. On the other hand, hypo-osmotic swelling response in these sperm went down drastically from 72 to 46% immediately after immobilization and there was no response at all after 60 min. In contrast, when Triton-x-100 (0.01%), a known spermicidal agent, was used in place of H(2)O(2), the immediate loss of sperm motility corresponded simultaneously with the loss of the other two functional parameters. Further, H(2)O(2) immobilized sperm revealed reduced (25-30%) nuclear chromatin decondensation as compared to 70% for the controls. A composition containing 2% H(2)O(2) in 0.9% NaCl was tested in rats as a vaginal contraceptive, depicting 100% efficacy in mating studies after 2 h of vaginal application. The findings indicate the potential of developing a water-based vaginal contraceptive using H(2)O(2) at an optimal concentration as utilized in the present study.

Cyclosporine affects motility and viability of human sperm when incubated together in vitro. Sperm motility was almost reduced to nil following 10 min of incubation with cyclosporine at a concentration of 1 mg/mL. However, 200 micrograms/mL of the drug has no effect on motility and viability when tested for up to 60 min under standard laboratory conditions. Cyclosporine effect on sperm was both dose and time dependent. Sperm sensitivity and susceptibility to cyclosporine even to lower doses increased significantly following withdrawal of bovine serum albumin from the incubating medium. Compared to untreated controls, lactate dehydrogenase was estimated higher by more than 2 to 4 times in the sperm-free incubating media, suggesting an altered membrane porosity in the affected spermatozoa.