Ka-mi-kae-kyuk-tang oriental herbal cocktail attenuates cyclophosphamide-induced leukopenia side effects in mouse

Ka-mi-kae-kyuk-tang (KMKKT) is an Oriental herbal medicinal cocktail and has been shown to have potent antiangiogenic, anticancer, and antimetastatic activities in preclinical animal models without observable side effects. We previously found that in prostate cancer xenograft experiments, treating tumor-bearing mice with KMKKT alleviated the body weight loss toward the end of the study, suggesting a general health-promoting activity. We investigated whether KMKKT alleviated cancer chemotherapy drug-induced leukopenia and other hematotoxicity in vivo using a mouse model. KMKKT was given once daily orally for 10 days to the mice before they were given cyclophosphamide (CPA) daily injection for 4 days. KMKKT blunted CPA-induced decrease in red blood cells, hemoglobin content, and the total white blood cell/leukocyte counts. Examination of the multiple organ sites involved in hematopoiesis, and lymphocyte differentiation and maturation showed the attenuated changes induced by CPA in each and every type of cells examined. Particularly, some of the cell types are fully restored in the bone marrow and even overstimulated in the Sca-1(+), CD117(+), or Sca1(+)/CD117(+) and CD34(+)/CD117(+) stem cells, supporting a role of KMKKT to stimulate hematopoietic stem cell (HSC) signaling to compensate for CPA-induced destruction of leukocytes and other cell types. Taken together, KMKKT might be a safe and effective herbal complementary and alternative medicine (CAM) modality to alleviate cancer drug-induced hematological side effects in addition to its anticancer activities. Preclinical investigations with other chemo- and radiation modalities are warranted to support planning translation consideration for human patients.     

Pathogenesis of pneumococcal infection

The pathogenesis of pneumococcal infection is a complex interplay between pneumococcal virulence determinants and the host immune response. Molecular studies have considerably advanced our knowledge and understanding of the precise structures and functions of the different determinants and their pathogenic roles. This review describes the mechanisms by which pneumococci attach, invade, evade lung defences and cause severe disease. Better understanding of the critical steps in this complex process will enable more effective clinical intervention to be developed to reduce the mortality exacted by this versatile pathogen.     

Protection against pneumococcal pneumonia in mice by monoclonal antibodies to pneumolysin

Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (P = 0.0288) or nontreated mice (P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.     

Reactivation of latent Herpes simplex virus after pneumococcal pneumonia in mice.

In attempts to reactivate latent herpes simplex virus, we instilled Diplococcus pneumoniae intratracheally into mice harboring latent infections in sacrosciatic spinal ganglia. All mice developed a severe pneumonia within 24 h and were given penicillin therapy. Representative mice that survived the penumonia were sacrificed at daily intervals, and appropriate tissues were examined for evidence of viral reactivation. Herpes simplex virus was reactivated in the ganglia and appeared to travel both proximally and distally in associated nerve trunks. Clinically apparent disease due to the virus was not detected in any mice.     

Stobadine-inhibitor of cyclophosphamide-induced micronuclei in mice

The potential antimutagenic effect of stobadine dipalmitate(STB) on the frequency of micronuclei in reticulocytes of peripheralblood in female ICR mice was studied. The cyclophosphamide modelwas used to verify this effect. Stobadine dipalmitate was administeredorally in three concentrations: STB I, 7.07; STB II, 23.6; STBIII, 70.07 mg/kg body wt 2 h prior to or 4 h after (STB II only)cyclophosphamide administration (intraperitoneally, twice 80mg/kg body wt with a 24 h interval). The method designed byHayashi et al. [(1990) Mutat. Res., 245, 245–249] wasused to prepare and to stain the slides. The results of theexperiment show that pretreatment with stobadine 2 h prior tocyclophosphamide administration significantly decreased itsmutagenic effect, as manifested by the reduced frequency ofmicronucleated reticulocytes. This protective effect of stobadinewas concentration-dependent with the highest concentration ofstobadine inducing the most pronounced decrease of micronuclei.Analysis and identification of the exact mechanism of the protectiveeffects of stobadine is the aim of our further studies. ¹To whom correspondence should be addressed     

Influence of neutropenia on the course of serotype 8 pneumococcal pneumonia in mice

Polymorphoneutrophils (PMNs) are important effector cells in host defense against pneumonia. However, PMNs can also induce inflammation and tissue damage. To investigate the contribution of PMNs to host defense against pneumococcal pneumonia, we determined the effect of the PMN-depleting rat monoclonal antibody RB6-8C5 (RB6) on survival and inflammatory and cellular response in the lungs to a lethal intranasal infection with a serotype 8 pneumococcus in BALB/c mice. Control mice received rat immunoglobulin G (rIgG). Strikingly, the survival of RB6-treated mice was significantly prolonged compared to that of rIgG-treated mice. Although the numbers of CFU in the lungs were statistically similar in both groups 4, 24, and 32 h after infection, rIgG-treated mice developed higher levels of bacteremia, and histopathological examination of the lungs of infected mice revealed marked differences between RB6- and rIgG-treated mice. RB6-treated mice had focal, perivascular lesions without accompanying parenchymal inflammation, and rIgG-treated mice had diffuse, interstitial parenchymal inflammation. Lung homogenates from the rIgG-treated mice had more leukocytes and significantly more total and apoptotic PMNs as determined by fluorescence-activated cell sorter analysis with Annexin V and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining of lung tissue samples. Studies with a pneumolysin-deficient mutant of the serotype 8 strain we used also demonstrated the prolonged survival of RB6- compared to rIgG-treated mice. Taken together, our findings suggest that PMNs enhance the likelihood of early death and alter the pathological response to pneumococcal lung infection in BALB/c mice with serotype 8 pneumonia without significantly affecting bacterial clearance or the cytokine response.     

Role of tumor necrosis factor alpha in pathogenesis of pneumococcal pneumonia in mice.

The production and role of tumor necrosis factor alpha (TNF-alpha) in pneumococcal pneumonia were investigated in a mouse pneumonia model. When approximately 10(6) CFU of Streptococcus pneumoniae TUM19 were used to inoculate CBA/J mice intranasally, TNF-alpha levels in the lungs and serum began to increase from 1 and 3 days after infection, respectively, concomitantly with the increase in bacterial counts in the lungs. Anti-TNF-alpha antibody accelerated bacterial proliferation in the blood and the death of the mice. Although serum levels of immunoglobulin G antibody against the infecting bacteria were not affected by the anti-TNF-alpha antibody treatment, neutrophil counts in the blood were decreased by the treatment. These results suggest that TNF-alpha produced in the course of pneumococcal pneumonia prevents bacteremia by increasing the number of neutrophils in the blood.     

Pathogenesis and pathophysiology of pneumococcal meningitis

Pneumococcal meningitis continues to be associated with high rates of mortality and long-term neurological sequelae. The most common route of infection starts by nasopharyngeal colonization by Streptococcus pneumoniae, which must avoid mucosal entrapment and evade the host immune system after local activation. During invasive disease, pneumococcal epithelial adhesion is followed by bloodstream invasion and activation of the complement and coagulation systems. The release of inflammatory mediators facilitates pneumococcal crossing of the blood-brain barrier into the brain, where the bacteria multiply freely and trigger activation of circulating antigen-presenting cells and resident microglial cells. The resulting massive inflammation leads to further neutrophil recruitment and inflammation, resulting in the well-known features of bacterial meningitis, including cerebrospinal fluid pleocytosis, cochlear damage, cerebral edema, hydrocephalus, and cerebrovascular complications. Experimental animal models continue to further our understanding of the pathophysiology of pneumococcal meningitis and provide the platform for the development of new adjuvant treatments and antimicrobial therapy. This review discusses the most recent views on the pathophysiology of pneumococcal meningitis, as well as potential targets for (adjunctive) therapy.     

The importance of neutrophils in resistance to pneumococcal pneumonia in adult and neonatal mice

Neonatal mice succumbed to intranasally-inoculatedStreptococcus pneumoniae doses which were as much as 250 times less than the doses that adult mice were resistant to. Neutrophil migration into lungs of neonates was similar in kinetics and intensity to that in adults in response to lethal doses ofS. pneumoniae. Interestingly, neutrophil infiltration into the lung alveoli of neonates occured at lower doses of bacteria than that required for similar responses in adults. Furthermore, depletion of neutrophils in adult and neonatal mice inoculated with low doses of bacteria resulted in significantly higher lung burdens of bacteria in neonatal mice as compared to adults. These data indicate that increased susceptibility of neonates toS. pneumoniae is not the result of incompletely developed neutrophil function and infact, indicate that neutrophils contribute more to resistance to low doses ofS. pneumoniae in neonates than they do in adult mice.     

ClpP粘膜免疫预防小鼠发生肺炎链球菌性肺炎和败血症

目的:肺炎链球菌毒力蛋白ClpP鼻腔粘膜免疫BALB/c小鼠,探讨其作为肺炎链球菌候选蛋白疫苗的价值.方法:利用制备的ClpP多克隆抗体,分析ClpP 在肺炎链球菌表面表达情况;ClpP蛋白粘膜免疫小鼠,TIGR4型肺炎链球菌分别经腹腔和鼻腔攻击,分析肺炎链球菌小鼠肺内定植情况并监测小鼠生存时间.结果:流式细胞技术证实了TIGR4肺炎链球菌表面表达ClpP毒力蛋白,粘膜免疫ClpP可以特异产生系统性和粘膜性的ClpP抗体,并可减少TIGR4肺炎链球菌在小鼠肺内的定植,延长小鼠生存时间.结论:ClpP粘膜免疫可预防小鼠发生肺炎链球菌性肺炎和败血症,进一步证实ClpP蛋白可作为肺炎链球菌的候选蛋白疫苗.     

Treatment with broadly neutralizing influenza antibodies reduces severity of secondary pneumococcal pneumonia in mice

Secondary bacterial pneumonia is a frequent complication of influenza, associated with high morbidity and mortality. We hypothesized that treatment with neutralizing influenza A antibody AT10_002 protects against severe secondary pneumococcal infection in a mouse model of influenza A infection. Influenza A (H3N2) virus–infected male C57Bl6 mice were treated intravenously with either AT10_002 or a control 2 days postinfection. Seven days later, both groups were infected with Streptococcus pneumoniae and killed 18 hours later. Mice receiving AT10_002 showed less loss of bodyweight compared with controls (+1% vs ?12%, P < .001), lower viral loads in bronchoalveolar lavage fluids (BALFs) (7 vs 194 RNA copies per µL; P < .001), and reduced bacterial outgrowth in lung homogenates (3.3 × 10¹ vs 2.5 × 10⁵ colony‐forming units per mg; P < .001). The treatment group showed lower pulmonary wet weights, lower cell counts, and lower protein levels in BALF compared with controls. Treatment with AT10_002 was associated with lower levels of tumor necrosis factor‐α, interleukin (IL)‐6, cytokine‐induced neutrophil chemoattractant (KC), and interferon‐γ in BALF and lower IL‐6 and KC in lung homogenates. Treatment with anti‐influenza antibody AT10_002 is associated with reduced weight loss, viral load, bacterial outgrowth, and lung injury in a murine model of secondary pneumococcal pneumonia following influenza infection.     

Complement levels in pneumococcal pneumonia.

Levels of complement proteins and functional activity of the alternate complement pathway were assessed in 39 patients with pneumococcal pneumonia. Mean levels of C3 and properdin and the functional activity of the alternate pathway in acute sera were significantly (P less than 0.05) below normal, whereas levels of components of the early classical pathway were normal. Although levels of factor B were in the normal range, they correlated significantly with C3 levels; there was no significant relation between C3 levels and C4 or C1q levels. The 19 patients iwth pneumococcal pneumonia and bacteremia had significantly lower mean values of properdin and factor B than the 20 patients without bacteremia, suggesting a more severe depression of the alternate complement pathway with bacteremia. During convalescence, complement levels were normal or elevated in most of the patients, but mean levels of properdin remained significantly below normal in bacteremic patients. Functional activity of the alternate pathway also remained below normal. These results indicate that there is a selective depression of the alternate pathway in patients with pneumococcal pneumonia, and they are consistent with the concept that the alternate pathway has an important role in host defenses in pneumococcal infection.     

Helminth infections predispose mice to pneumococcal pneumonia but not to other pneumonic pathogens

Pneumonia is the leading killer of children worldwide. Here, we report that helminth-infected mice develop fatal pneumonia when challenged with Streptococcus pneumoniae. Mice were chronically infected with either the flatworm Taenia crassiceps or the roundworm Heligmosomoides polygyrus. Upon challenge with a pneumonic type 3 strain of S. pneumoniae (A66.1), the worm-infected mice developed pneumonia at a rate and to a degree higher than age-matched control mice as measured by bioluminescent imaging and lung titers. This predisposition to pneumonia appears to be specific to S. pneumoniae, as worm-infected mice did not show evidence of increased morbidity when challenged with a lethal dose of influenza virus or sublethal doses of Staphylococcus aureus or Listeria monocytogenes. The defect was also present when worm-infected mice were challenged with a type 2 sepsis-causing strain (D39); an increased rate of pneumonia, decreased survival, and increased lung and blood titers were found. Pneumococcal colonization and immunity against acute otitis media were unaffected. Anti-helminthic treatment in the H. polygyrus model reversed this susceptibility. We conclude that helminth coinfection predisposes mice to fatal pneumococcal pneumonia by promoting increased outgrowth of bacteria in the lungs and blood. These data have broad implications for the prevention and treatment for pneumonia in the developing world, where helminth infections are endemic and pneumococcal pneumonia is common.     

Complement-fixing antibody response in pneumococcal pneumonia.

Previous studies of complement-fixing antibodies to pneumococcal capsular polysaccharides in humans have yielded conflicting results. We studied 65 sera from 25 patients with pneumococcal pneumonia, using both fresh sera and heat-inactivated sera with added human complement. Only 4 of the 25 patients developed detectable levels of complement-fixing anticapsular antibody. Of the 25 patients, 22 developed detectable levels of hemagglutinating anticapsular antibody, indicating that they were able to develop an immunological response during the infection. Most of the antibody detected by hemagglutination was sensitive to 2-mercaptoethanol, but some 2-mercaptoethanol-resistant antibody was also detected. In studies with rabbit antiserum, the complement fixation test was found to be as sensitive as the hemagglutination test for detection of anticapsular antibody. It is not clear why detectable levels of complement-fixing antibody do not develop more often in patients with pneumococcal pneumonia. Studies of purified anticapsular antibody would be of interest to determine whether or not these antibodies are restricted to immunoglobulin subclasses having a limited capacity to fix complement.     

Pathogenesis of infant pneumonia]

Basing on morphofunctional study of 101 autopsy cases of children under one year of age, the role of premorbid background, congenital and acquired immunodeficiencies is shown in the onset of pneumonia. The scheme of the pneumonia development is suggested.     

Leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia

The adipocyte-derived hormone leptin is an important regulator of appetite and energy expenditure and is now appreciated for its ability to control innate and adaptive immune responses. We have reported previously that the leptin-deficient ob/ob mouse exhibited increased susceptibility to the Gram-negative bacterium Klebsiella pneumoniae. In this report we assessed the impact of chronic leptin deficiency, using ob/ob mice, on pneumococcal pneumonia and examined whether restoring circulating leptin to physiological levels in vivo could improve host defences against this pathogen. We observed that ob/ob mice, compared with wild-type (WT) animals, exhibited enhanced lethality and reduced pulmonary bacterial clearance following Streptococcus pneumoniae challenge. These impairments in host defence in ob/ob mice were associated with elevated levels of lung tumour necrosis factor (TNF)-alpha, macrophage inflammatory peptide (MIP)-2 [correction added after online publication 28 September 2007: definition of MIP corrected], prostaglandin E(2) (PGE(2)), lung neutrophil polymorphonuclear leukocyte (PMN) counts, defective alveolar macrophage (AM) phagocytosis and PMN killing of S. pneumoniae in vitro. Exogenous leptin administration to ob/ob mice in vivo improved survival and greatly improved pulmonary bacterial clearance, reduced bacteraemia, reconstituted AM phagocytosis and PMN H(2)O(2) production and killing of S. pneumoniae in vitro. Our results demonstrate, for the first time, that leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia. Further investigations are warranted to determine whether there is a potential therapeutic role for this adipokine in immunocompromised patients.     

Cost effectiveness of vaccination against pneumococcal pneumonia

We used cost-effectiveness analysis to examine the medical-care costs of vaccination against pneumococcal pneumonia in relation to its effects on health. Vaccination could add a year of healthy life among all age groups for about $4,800 in net medical-care costs. Cost-effectiveness ratios vary according to the age of the person vaccinated--from $1,000 per year of healthy life for an adult 65 years old or older to $77,000 per year of healthy life for a child between the ages of two and four. These ratios may change substantially with variations in such factors as the cost of vaccination, the duration of immunity, the efficacy and composition of the vaccine, and the percentage of pneumonia that is pneumococcal. This analysis has particular relevance for the Medicare program, since present legislation excludes coverage of most immunizations and other preventive services. Provision of pneumococcal vaccine to the elderly and inclusion of the vaccine as a Medicare benefit merit serious consideration.     

False no-growth blood cultures in pneumococcal pneumonia.

The growth of Streptococcus pneumoniae in commercial media containing 14C-labeled substrates was studied experimentally; the results of blood cultures that were positive for S. pneumoniae over a 14-month period were analyzed to explain no-growth but radiometrically positive blood cultures from four patients with clinically diagnosed pneumococcal pneumonia. The growth of S. pneumonoiae in aerobic blood culture vials resulted in a chocolate color in the medium. S. pneumoniae grew rapidly in both aerobic and anaerobic media, but 14CO2 evolved from the metabolism of the labeled substrates was detected only in the aerobic culture vials. Radiometric detection lagged behind growth of the organisms and was accompanied by visual changes in the media. By 24 h, the viability of the culture was on the decline; radiometric readings remained positive even when the culture had died.     

Monocyte recruitment into the lungs in pneumococcal pneumonia

The recruitment of monocytes into the alveolar spaces is crucial for clearing infections and resolving the inflammatory response. We have previously reported the effect of acute pneumonia on monocyte transport through the bone marrow, and the present study concerns their clearance from the blood and migration into the lung airspaces. Dividing monocytes were labeled with the thymidine analog, 5'-bromo-2'-deoxyuridine (BrdU). Whole blood containing the labeled monocytes (MO(BrdU)) was transfused from either donor rabbits with pneumonia or from uninfected controls into recipients with pneumonia, where they were detected in blood and tissues using a double immunostaining method. The results show that MO(BrdU) from infected animals rapidly disappeared from the circulation (P < 0.05), preferentially sequestered in the infected lung tissue within 1 h (22.0 +/- 3.3% versus 6.0 +/- 0.4%, pneumonic region versus peripheral blood, P < 0.05), and accumulated to a greater degree in pneumonic airspaces than control monocytes 48 h after transfusion (3.9 +/- 0.5% versus 1.1 +/- 0.1%, P < 0.05). We conclude that immature monocytes released from the marrow by pneumonia sequester rapidly in lung microvessels but their migration in pneumonic airspaces is delayed.