Fluorescence lifetime imaging and phasor analysis of intracellular porphyrinic photosensitizers applied with different polymeric formulations.

The fluorescence lifetime of a porphyrinic photosensitizer (PS) is an important parameter to assess the aggregation state of the PS even in complex biological environments. Aggregation-induced quenching of the PS can significantly reduce the yield of singlet oxygen generation and thus its efficiency as a medical drug in photodynamic therapy (PDT) of diseased tissues. Hydrophobicity and the tendency to form aggregates pose challenges on the development of efficient PSs and often require carrier systems. A systematic study was performed to probe the impact of PS structure and encapsulation into polymeric carriers on the fluorescence lifetime in solution and in the intracellular environment. Five different porphyrinic PSs including chlorin e6 (Ce6) derivatives and tetrakis(m-hydroxyphenyl)-porphyrin and -chlorin were studied in free form and combined with polyvinylpyrrolidone (PVP) or micelles composed of triblock-copolymers or Cremophor. Following incubation of HeLa cells with these systems, fluorescence lifetime imaging combined with phasor analysis and image segmentation was applied to study the lifetime distribution in the intracellular surrounding. The data suggest that for free PSs, the structure-dependent cell uptake pathways determine their state and emission lifetimes. PS localization in the plasma membrane yielded mostly monomers with long fluorescence lifetimes whereas the endocytic pathway with subsequent lysosomal deposition adds a short-lived component for hydrophilic anionic PSs. Prolonged incubation times led to increasing contributions from short-lived components that derive from aggregates mainly localized in the cytoplasm. Encapsulation of PSs into polymeric carriers led to monomerization and mostly fluorescence emission decays with long fluorescence lifetimes in solution. However, the efficiency depended on the binding strength that was most pronounced for PVP. In the cellular environment, PVP was able to maintain monomeric long-lived species over prolonged incubation times. This was most pronounced for Ce6 derivatives with a logP value around 4.5. Micellar encapsulation led to faster release of the PSs resulting in multiple components with long and short fluorescence lifetimes. The hydrophilic hardly aggregating PS exhibited a mostly stable invariant lifetime distribution over time with both carriers. The presented data are expected to contribute to optimized PDT treatment protocols and improved PS-carrier design for preventing intracellular fluorescence quenching. In conclusion, amphiphilic and concurrent hydrophobic PSs with high membrane affinity as well as strong binding to the carrier have best prospects to maintain their photophysical properties in vivo and serve thus as efficient photodynamic diagnosis and PDT drugs.

Comparing PVP and Polymeric Micellar Formulations of a PEGylated Photosensitizing Phthalocyanine by NMR and Optical Techniques.

Phthalocyanines are ideal candidates as photosensitizers for photodynamic therapy (PDT) of cancer due to their favorable chemical and photophysical properties. However, their tendency to form aggregates in water reduces PDT efficacy and poses challenges in obtaining efficient forms of phthalocyanines for therapeutic applications. In the current work, polyvinylpyrrolidone (PVP) and micellar formulations were compared for encapsulating and monomerizing a water-soluble zinc phthalocyanine bearing four non-peripheral triethylene glycol chains (Pc1). 1H NMR spectroscopy combined with UV-vis absorption and fluorescence spectroscopy revealed that Pc1 exists as a mixture of regioisomers in monomeric form in dimethyl sulfoxide but forms dimers in an aqueous buffer. PVP, polyethylene glycol castor oil (Kolliphor RH40), and three different triblock copolymers with varying proportions of polyethylene and polypropylene glycol units (termed P188, P84, and F127) were tested as micellar carriers for Pc1. 1H NMR chemical shift analysis, diffusion-ordered spectroscopy, and 2D nuclear Overhauser enhancement spectroscopy was applied to monitor the encapsulation and localization of Pc1 at the polymer interface. Kolliphor RH40 and F127 micelles exhibited the highest affinity for encapsulating Pc1 in the micellar core and resulted in intense Pc1 fluorescence emission as well as efficient singlet oxygen formation along with PVP. Among the triblock copolymers, efficiency in binding and dimer dissolution decreased in the order F127 > P84 > P188. PVP was a strong binder for Pc1. However, Pc1 molecules are rather surface-attached and exist as monomer and dimer mixtures. The results demonstrate that NMR combined with optical spectroscopy offer powerful tools to assess parameters like drug binding, localization sites, and dynamic properties that play key roles in achieving high host-guest compatibility. With the corresponding adjustments, polymeric micelles can offer simple and easily accessible drug delivery systems optimizing phthalocyanines' properties as efficient photosensitizers.

Differential Effect of Community Rehabilitation Reform on Hospitalizations of Patients with Chronic Psychotic Disorders With and Without Substance Use Disorder, Israel, 1991-2016.

The co-occurrence of schizophrenia and substance use disorder (SUD) is clinically challenging and increasingly prevalent. This study compares trends in hospitalization characteristics of chronic psychotic patients with and without SUD in Israel, before and after introduction of the Community Rehabilitation of Persons with Mental Disability Law in 2000. The National Psychiatric Case Registry provided data on 18,684 adults with schizophrenia/schizoaffective disorders, hospitalized in 1991-2016 (at least once in 2010-2015). Repeated-measures ANOVA was used to measure the effect (and interactions) of group (patients with and without co-occurring disorders (COD)), time-period (Period1: 1991-2000, Period2: 2001-2009, Period3: 2010-2016) and age, on hospitalization measures-average length of stay (LOS), annual number of hospitalizations and hospitalization days. Among non-COD patients hospitalized in all three periods, LOS declined by half from 133.3 days in Period1 to 63.2 in Period3, and the annual number of hospitalizations increased slightly from 0.45 to 0.56. Among COD patients, LOS declined moderately from 82.7 days to 58.3 days, while annual hospitalizations increased dramatically from 0.56 to 0.82. The annual average number of hospitalization days/capita declined from 49.7 in Period1 to 26.3 in Period3 among non-COD patients, yet remained virtually unchanged among COD patients-39.6 and 37.4 in the two periods, respectively. Since introduction of the law, a significant improvement in hospitalization characteristics of chronic psychotic non-COD patients has been noted, whereas the situation worsened somewhat for COD patients. Community rehabilitation services for COD patients in Israel have yet to develop as a suitable alternative to hospitalization, and additional rehabilitation services are urgently needed.

Psychiatric Hospitalizations of Chronic Psychotic Disorder Patients With and Without Dual Diagnosis, Israel, 1963-2016.

Objective: A significant proportion of patients with severe mental illness also experience substance use disorder. For these dual diagnosis (DD) patients, treatment is more complicated and prognosis is worse. Despite the introduction of the Community Rehabilitation of Persons With Mental Health Disability Law in 2000 and ongoing national mental health reforms, psychiatric services in Israel are not meeting the needs of an increasing number of DD patients. This study examines, for the first time in Israel, the prevalence of DD and patterns of psychiatric hospitalizations of chronic psychotic disorder patients with and without substance use disorder. Methods: The National Psychiatric Case Registry provided data on 18,684 persons with schizophrenia/schizoaffective disorders, aged 18-65, with a psychiatric hospitalization during the period 1963-2016 (with at least one hospitalization in 2010-15). Patients were considered as having DD if their substance use disorder was indicated in at least two, or 20%, of hospitalizations. Regression modeling predicted hospitalization measures (number of hospitalizations, total days hospitalized, length of stay). Results were also analyzed by legal status of admission (voluntary or involuntary; psychiatrist-ordered and court-ordered). Results: One-third of patients with chronic psychotic disorder met DD criteria, with a threefold higher rate among males (37.1%) than females (12.8%). Particularly high rates of DD (nearly 50%) were noted among male immigrants from Ethiopia. Compared with non-substance use disorder patients, DD patients had a significantly younger mean age at first hospitalization and shorter average length of stay per hospitalization but a greater number of hospitalizations and total hospital days (p < .0001 for all comparisons). The associations between DD status and hospitalization characteristics remained significant even after accounting for the effects of confounding factors. Hospitalization characteristics were also associated significantly with sex, population group, age, age at first hospitalization, and country of origin. The rate of court-ordered observation or hospitalization was threefold higher in the DD group. Conclusions: These findings, which broadly align with other countries, reflect a scarcity of outpatient services for DD patients with schizophrenia/schizoaffective disorder and substance use disorder. To achieve long-term mental health improvements, an expansion of community-based integrative treatment and rehabilitation services is needed in Israel.

Does bovine besnoitiosis affect the sexual function of chronically infected bulls?

Bovine besnoitiosis is a reemerging disease in Europe. The clinically Besnoitia besnoiti infection in bulls is characterized by fever, nasal discharge, and orchitis in the acute phase and by scleroderma in the chronic phase. However, in many bulls, B besnoiti infection remains at a subclinical stage. Bull infertility is an economically relevant consequence of besnoitiosis infection. It is not clear, however, if semen quality returns to normal levels when infected animals have clinically recovered. The aim of this study was to examine the relationship between chronic besnoitiosis and bull sexual function in a region of eastern France, where the disease is reemerging, by comparing semen quality and genital lesions in 11 uninfected, 17 subclinically infected, and 12 clinically infected bulls. The presence of anti-B besnoiti antibodies was detected by Western blot test. Semen was collected by electroejaculation. Bulls clinically infected with B besnoiti showed significantly more genital tract alterations than uninfected or subclinically infected bulls. No relationship was evidenced between besnoitiosis infectious status and semen quality, whereas a significant relationship was noted between genital lesions and semen score. This means that in the absence of moderate to severe genital lesions, chronic bovine besnoitiosis is unlikely to alter semen quality. However, as the presence of infected animals could lead to spread of the disease, culling or separation of clinically infected bulls from the remaining healthy animals is strongly recommended.

Effect of gonadorelin, lecirelin, and buserelin on LH surge, ovulation, and progesterone in cattle.

Analogs of gonadoliberin (GnRH) are widely used in cattle to synchronize estrus and to induce ovulation, as well as for the treatment of ovarian cysts. The aim of this study was to compare the plasma profiles of LH and progesterone and the follicular dynamics in response to the administration of gonadorelin, lecirelin, or buserelin at the dose recommended to induce ovulation. In addition, the biological response to a half dose of lecirelin was assessed. Twelve healthy Holstein female cows were divided into four sequence groups, according to a Latin square design and received the four treatments during the four periods of the study. Before each period, the estrous cycle was synchronized, and on Day 6 or 7 of the ensuing cycle, the time at which it was most likely to have a dominant follicle, 100 µg of gonadorelin, 25 µg of lecirelin, 50 µg of lecirelin, or 10 µg of buserelin was administered to the cows. Blood samples were regularly collected for up to 4 days after the GnRH administrations. The plasma LH response was evaluated for up to 6 hours after administration, and the plasma progesterone response and ovarian follicular dynamics were evaluated for up to 4 days. There was a significantly lower LH release after gonadorelin treatment compared to lecirelin at the doses of 25 or 50 µg and the buserelin treatment. The mean maximal LH concentration after gonadorelin treatment was 2.5 lower than after lecirelin or buserelin treatment and was reached 1 hour earlier. Four days after the GnRH administration (i.e., at Days 10-11 of the estrous cycle), the overall mean increase in plasma progesterone concentration was 70% and did not differ between the treatment groups. The percentage of disappearance of the dominant follicle (84.8% of ovulation and 4.3% of luteinization) after GnRH treatment was high (73%, 82%, 100%, and 100%, for gonadorelin, lecirelin at the doses of 25 and 50 µg, and buserelin, respectively) and did not differ between the GnRH treatments. The follicle disappearance was followed by the emergence of a synchronous follicle wave within 2 days in almost all the heifers. Altogether, our data show that the three GnRH analogs, at the doses indicated for the induction of ovulation or at a half dose for lecirelin, are almost equally effective to induce the disappearance of the dominant follicle at Day 6 to 7 of the estrous cycle.

No detection of Besnoitia besnoiti DNA in the semen of chronically infected bulls.

Bovine besnoitiosis is a chronic and debilitating disease observed in many European countries that may cause important economic losses in cattle. The recent widespread of the parasite in Europe had led the European Food Safety Authority to declare bovine besnoitiosis as a re-emerging disease in Europe. Many aspects of the epidemiology of bovine besnoitiosis such as the main routes of transmission are still unclear and need to be further studied. Among the different hypotheses, a sexual transmission has not yet been investigated. Therefore, the aim of this study was to evaluate the presence of Besnoitia besnoiti DNA in the semen of naturally infected bulls by using a highly sensitive method (real-time qPCR). Both pre-sperm and sperm fractions of 40 bulls, including seronegative (n = 11), seropositive subclinically (n = 17), and seropositive clinically (n = 12) infected animals, were collected by electroejaculation and analyzed by real-time qPCR. No B. besnoiti DNA was detected in 27 pre-sperm and 28 sperm fractions of the 40 examined bulls, suggesting that the transmission of B. besnoiti infection by the semen of chronically infected bulls is very unlikely.