<p>Epilepsy is comorbid with cognitive and psychiatric dysfunctions. This pathophysiology, associated with hippocampal synaptodendritic structural and functional changes, is exacerbated by prolonged seizures (status epilepticus; SE). We found a correlation between hippocampal dendritic loss and microgliosis after SE, along with hyperactivation of the classical complement pathway (C1q-C3). These paralleled increased seizure frequency and memory deficits in a rat model of SE and acquired epilepsy. C1q leads to C3 cleavage into biologically active fragments C3a and C3b. Evidence suggests that C1q and C3b contribute to synaptic stripping by microglia in the developing brain and neurodegenerative disorders. Thus, we hypothesized that SE-induced C3 activation may alter hippocampal synaptic protein levels thereby promoting memory deficits. </p> <p>To test the hypothesis, different groups of wild type (WT) or C3 deficient (C3KO) mice were injected with pilocarpine (350mg/kg) to induce SE or saline (controls): WT-C, WT-SE, C3KO-C, and C3KOSE. At two weeks after SE, mice were subjected to novel object recognition (NOR) to evaluate recognition memory, and Barnes maze (BM) to measure hippocampal-dependent spatial learning and memory. Following behavioral testing, mice were sacrificed and hippocampi collected at either 2 or 5 weeks after SE to measure changes in C3 protein levels and levels of synaptic proteins including PSD95, Vglut1, and Vgat. As a method of verifying our findings, we used a second model of pilocarpine-induced SE in male Sprague Dawley rats. Starting at 7 days after SE, rats were treated with cobra venom factor (CVF; 100ng/g, i.p.) or vehicle (veh) every third day. On days 14-15 rats were subjected to open field and NOR to measure anxiety and recognition memory. On day 16, rats were sacrificed and hippocampi collected for western blotting.</p> <p>WT and C3KO mice were able to reach stage 4.5-6 seizures after pilocarpine injections. In NOR trial 1, exploration time for both objects was similar in all groups (<i>p</i> > .05). In trial 2, WT-C and C3KO-C mice spent more time exploring the novel object than the familiar one (<i>p</i> < .05) while WT-SE mice explored both objects equally (<i>p</i> > .05). Interestingly, C3KO-SE mice spent more time with the novel object similar to controls (<i>p</i> > .05), suggesting that the deficit in object recognition memory induced by SE was attenuated in C3KO mice. Similarly, veh- and CVF-treated control rats spent more time exploring the novel object during trial 2 (<i>p</i> < .05). The veh-treated SE rats did not show significant preference for the novel object versus familiar (<i>p</i> > .05), whereas the CVF-treated SE rats explored the novel object significantly more than the familiar (p < .05). These findings support that C3 inhibition after SE prevents recognition memory deficits. Furthermore, there was a reduction in synaptic proteins PSD95 and Vgat in the SE-veh group compared to the C-veh group. This difference was not observed in the C-CVF and SE-CVF groups, suggesting that blocking C3 after SE is neuroprotective against hippocampal synaptic loss.</p> <p>Taken together, these findings are the first to show an association between C3 activation and hippocampal and cognitive deficits in two rodent models of SE and acquired TLE. We found that depletion of C3 is sufficient to attenuate SE-induced deficits in NOR-evaluated recognition memory and changes in the levels of an inhibitory synaptic protein. In conclusion, our data suggest that SE-induced complement C3 activation contributes to hippocampal synaptic remodeling and impairments in recognition memory, and that the complement C3 may be a potential therapeutic target for the memory comorbidities associated with SE. Future studies will determine the effect of C3 inhibition on spontaneous recurrent seizures, and whether C3-guided and microglial-dependent phagocytosis is an underlying mechanism for the SE-induced epileptogenic synaptic remodeling.</p>

Objetivos: Os objetivos deste estudo foram: avaliar a frequência de imunodeficiências primárias de anticorpos e Complemento em pacientes com lupus eritematoso sistêmico juvenil (LESJ); avaliar possíveis associações entre a presença de imunodeficiência primária (IDP) e dados demográficos, ocorrência de infecções, manifestações clínicas, atividade da doença, dano cumulativo e terapêutica direcionada ao LESJ; e determinar a frequência do anticorpo anti-C1q, estabelecendo a sua especificidade, sensibilidade e valores preditivos para o diagnóstico de LESJ. Métodos: Setenta e dois pacientes com LESJ foram avaliados para a determinação dos níveis séricos de imunoglobulinas (IgG, IgA, IgM e IgE) e subclasses de IgG, e dos componentes iniciais da via clássica do sistema Complemento (C1q, C1r, C1s, C4, C2, C3). Sessenta e sete pacientes e 26 controles saudáveis foram avaliados para a presença do anticorpo anti-C1q. O número de cópias do gene C4 foi determinado por PCR (reação de polimerase em cadeia) em tempo real nos pacientes com deficiência de C4. Setenta pacientes foram avaliados para a presença de deficiência de C2 tipo I. Resultados: Evidência de IDP foi identificada em 16 pacientes (22%): 3 com deficiência (D) de C2, 3 com C4D, 2 com C1qD, 4 com IgG2D (<20mg/dL), 3 com IgAD (<7mg/dL), e 3 com IgMD (<35mg/dL); um destes pacientes apresentou deficiência concomitante de IgA, C4 e C2. Quatro dos 13 pacientes do sexo masculino (30%) e 12 das 59 pacientes do sexo feminino (20%) apresentaram diagnóstico de IDP. As características clínicas de LES não diferiram entre os pacientes com e sem IDP. A mediana do SLICC/ACR-DI foi maior entre os pacientes com IDP (p=0,0033), assim como a frequência de SLICC/ACR-DI>1 (p=0,023). Os grupos também foram semelhantes quanto à ocorrência de infecção e terapêutica utilizada para o LESJ. Os únicos dois casos de LESJ com idade de início antes dos 2 anos apresentaram C1qD e IgMD, respectivamente. Para o diagnóstico de LESJ, o anticorpo anti-C1q apresentou especificidade de 100% (IC 86.7-100%), sensibilidade de 19.4% (IC 10.7-30.8%), valor preditivo positivo de 100% (IC 75.3-100%) e valor preditivo negativo de 32,5% (IC 22,4-43,9%). Conclusões: Foi observada uma elevada frequência de imunodeficiências de anticorpos e Complemento nos pacientes com LESJ, sugerindo que esses defeitos podem contribuir para a patogênese do lúpus. Esses achados indicam que os dois grupos de IDPs devem ser investigados em pacientes com LES de início precoce e de maior gravidade / Objectives. The objectives of this study were: to establish the frequency of primary immunoglobulin and Complement deficiency in Juvenile SLE (JSLE); to evaluate possible associations between the presence of primary immunodeficiency and demographic data, occurrence of infections, JSLE clinical manifestations, disease activity, cumulative damage and therapy; and to determine the frequency of anti-C1q antibody, establishing its sensitivity, specificity and predictive values for JSLE diagnosis. Methods. Seventy-two JSLE patients were analyzed for serum levels of immunoglobulin classes (IgG, IgA, IgM e IgE) and IgG subclasses and early components of the classical Complement pathway (C1q, C1r, C1s, C4, C2, C3). Sixty-seven patients and 26 healthy controls were evaluated for the presence of anti-C1q antibody. C4 gene copy number was determined by real time PCR (polymerase chain reaction) in C4 deficient patients. Seventy patients were analyzed by PCR for the presence of type I C2 deficiency. Results. Evidence of PID was identified in 16 patients (22%): 3 with C2 deficiency (D), 3 with C4D, 2 with C1qD, 4 with IgG2D (<20mg/dL), 3 with IgAD (<7mg/dL), and 3 with IgMD (<35mg/dL); one of these patients presented concomitant IgA, C2 and C4 deficiency. Four out of the 13 boys (30%) and 12 out of 59 girls (20%) had PID diagnosis. SLE features did not differ between patients with and without PID. The median SLICC/ACR-DI was higher among PID subjects (p=0.0033), as was the frequency of SLICC/ACR-DI>1 (p=0.023). Both groups did not differ regarding the occurrence of infections and therapeutic for JSLE. The only 2 cases with age of onset below 2 years presented C1qD and IgMD, respectively. For JSLE diagnosis, the anti-C1q antibodies presented a specificity of 100% (CI 86.7-100%), sensitivity of 19.4% (CI 10.7-30.8%), positive predictive value of 100% (CI 75.3-100%) and negative predictive value of 32,5% (CI 22,4-43,9%). Conclusions. A high frequency of immunoglobulin and Complement deficiency was observed in this JSLE series, suggesting that these defects may contribute to lupus development. Our findings indicate that these two groups of PID should be investigated in early-onset and severe lupus

Objetivos: Os objetivos deste estudo foram: avaliar a frequência de imunodeficiências primárias de anticorpos e Complemento em pacientes com lupus eritematoso sistêmico juvenil (LESJ); avaliar possíveis associações entre a presença de imunodeficiência primária (IDP) e dados demográficos, ocorrência de infecções, manifestações clínicas, atividade da doença, dano cumulativo e terapêutica direcionada ao LESJ; e determinar a frequência do anticorpo anti-C1q, estabelecendo a sua especificidade, sensibilidade e valores preditivos para o diagnóstico de LESJ. Métodos: Setenta e dois pacientes com LESJ foram avaliados para a determinação dos níveis séricos de imunoglobulinas (IgG, IgA, IgM e IgE) e subclasses de IgG, e dos componentes iniciais da via clássica do sistema Complemento (C1q, C1r, C1s, C4, C2, C3). Sessenta e sete pacientes e 26 controles saudáveis foram avaliados para a presença do anticorpo anti-C1q. O número de cópias do gene C4 foi determinado por PCR (reação de polimerase em cadeia) em tempo real nos pacientes com deficiência de C4. Setenta pacientes foram avaliados para a presença de deficiência de C2 tipo I. Resultados: Evidência de IDP foi identificada em 16 pacientes (22%): 3 com deficiência (D) de C2, 3 com C4D, 2 com C1qD, 4 com IgG2D (<20mg/dL), 3 com IgAD (<7mg/dL), e 3 com IgMD (<35mg/dL); um destes pacientes apresentou deficiência concomitante de IgA, C4 e C2. Quatro dos 13 pacientes do sexo masculino (30%) e 12 das 59 pacientes do sexo feminino (20%) apresentaram diagnóstico de IDP. As características clínicas de LES não diferiram entre os pacientes com e sem IDP. A mediana do SLICC/ACR-DI foi maior entre os pacientes com IDP (p=0,0033), assim como a frequência de SLICC/ACR-DI>1 (p=0,023). Os grupos também foram semelhantes quanto à ocorrência de infecção e terapêutica utilizada para o LESJ. Os únicos dois casos de LESJ com idade de início antes dos 2 anos apresentaram C1qD e IgMD, respectivamente. Para o diagnóstico de LESJ, o anticorpo anti-C1q apresentou especificidade de 100% (IC 86.7-100%), sensibilidade de 19.4% (IC 10.7-30.8%), valor preditivo positivo de 100% (IC 75.3-100%) e valor preditivo negativo de 32,5% (IC 22,4-43,9%). Conclusões: Foi observada uma elevada frequência de imunodeficiências de anticorpos e Complemento nos pacientes com LESJ, sugerindo que esses defeitos podem contribuir para a patogênese do lúpus. Esses achados indicam que os dois grupos de IDPs devem ser investigados em pacientes com LES de início precoce e de maior gravidade / Objectives. The objectives of this study were: to establish the frequency of primary immunoglobulin and Complement deficiency in Juvenile SLE (JSLE); to evaluate possible associations between the presence of primary immunodeficiency and demographic data, occurrence of infections, JSLE clinical manifestations, disease activity, cumulative damage and therapy; and to determine the frequency of anti-C1q antibody, establishing its sensitivity, specificity and predictive values for JSLE diagnosis. Methods. Seventy-two JSLE patients were analyzed for serum levels of immunoglobulin classes (IgG, IgA, IgM e IgE) and IgG subclasses and early components of the classical Complement pathway (C1q, C1r, C1s, C4, C2, C3). Sixty-seven patients and 26 healthy controls were evaluated for the presence of anti-C1q antibody. C4 gene copy number was determined by real time PCR (polymerase chain reaction) in C4 deficient patients. Seventy patients were analyzed by PCR for the presence of type I C2 deficiency. Results. Evidence of PID was identified in 16 patients (22%): 3 with C2 deficiency (D), 3 with C4D, 2 with C1qD, 4 with IgG2D (<20mg/dL), 3 with IgAD (<7mg/dL), and 3 with IgMD (<35mg/dL); one of these patients presented concomitant IgA, C2 and C4 deficiency. Four out of the 13 boys (30%) and 12 out of 59 girls (20%) had PID diagnosis. SLE features did not differ between patients with and without PID. The median SLICC/ACR-DI was higher among PID subjects (p=0.0033), as was the frequency of SLICC/ACR-DI>1 (p=0.023). Both groups did not differ regarding the occurrence of infections and therapeutic for JSLE. The only 2 cases with age of onset below 2 years presented C1qD and IgMD, respectively. For JSLE diagnosis, the anti-C1q antibodies presented a specificity of 100% (CI 86.7-100%), sensitivity of 19.4% (CI 10.7-30.8%), positive predictive value of 100% (CI 75.3-100%) and negative predictive value of 32,5% (CI 22,4-43,9%). Conclusions. A high frequency of immunoglobulin and Complement deficiency was observed in this JSLE series, suggesting that these defects may contribute to lupus development. Our findings indicate that these two groups of PID should be investigated in early-onset and severe lupus

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.